Methods for Identifying and Compounds Useful for Increasing the Functional Activity and Cell Surface Expression of CF-Associated Mutant Cystic Fibrosis Transmembrance Conductance Regulator

ABSTRACT

The present invention relates to agents, and methods for identifying compounds, which agents and compounds result in the modulation of cellular trafficking of proteins in particular that of CF-associated mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In addition, the invention relates to compositions and methods for the use thereof in treating conditions that are characterized by an ER-associated protein misfolding and abnormal cellular trafficking of disease-associated proteins, including cystic fibrosis (CF).

BACKGROUND OF THE INVENTION

The present invention relates to agents, and methods for identifyingcompounds, which agents and compounds result in the increased functionalactivity of CF-associated mutant Cystic Fibrosis TransmembraneConductance Regulator (CFTR). In addition, the invention relates tocompositions and methods for the use thereof in treating conditions thatare characterized by a decrease in function of CF-associated mutant CFTRincluding cystic fibrosis (CF), and other protein misfolding diseases.

Cystic Fibrosis Transmembrane Conductance Regulator, a member of theATP-binding cassette (ABC) transporter family, is believed to regulatethe chloride channel responsible for cAMP-mediated chloride secretion inepithelial cells. For reviews on cystic fibrosis we refer to Guggino andStanton, 2006) and Rowe et al., 2005. By its chloride channel function,CFTR plays a key role in chloride secretion and water balance inepithelia throughout the body. CFTR has been identified and sequenced(Riordan et al., 1989). Defects in this gene causing diminished activityand/or expression of CFTR lead to cystic fibrosis. CF is the most commonfatal genetic disease in humans affecting approximately one in every2,500 infants born in the United States of America. In patients with CF,expression of the CF-associated gene in epithelial cells leads toreduced cellular apical chloride conductance, causing an imbalance inion and fluid transport. It is widely believed that this leads to theabnormal mucus secretion in pancreatic ductules and in the airways thatultimately results in the pulmonary infections and epithelial celldamage typically associated with disease progression in CF. In additionto respiratory problems, CF patients typically suffer fromgastrointestinal problems, and pancreatic insufficiency. Males arealmost uniformly infertile and fertility is decreased in females.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease-causing mutations. At present, more than 1000mutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/ orhttp://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602421), butpopulation studies have indicated that the most common CF mutation, adeletion of the 3 nucleotides that encode phenylalanine at position 508of the CFTR amino acid sequence, is associated with approximately 70% ofthe cases of cystic fibrosis. The mutated CFTR protein is referred to asΔF508.

It is believed that the deletion of residue 508 in ΔF508-CFTR preventsthe nascent protein from folding correctly, resulting in the inabilityof this mutant protein to exit the endoplasmic reticulum (ER), andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced (Quinton, 1990).Studies have shown, however, that ΔF508-CFTR, when presented at theplasma membrane is functional as a cAMP-responsive Cl⁻ channel (Denninget al., 1992). Correcting ΔF508-CFTR maturation, allowing exit ofΔF508-CFTR from the ER, or enhancing the activity of ΔF508-CFTR wouldconstitute a mode of action of a novel drug to treat CF.

In fact, the cellular phenomenon of defective ER processing of ABCtransporters, or other proteins, by the ER machinery has been shown tobe the underlying basis not only for CF disease, but for a wide range ofother isolated and inherited diseases (Ulloa-Aguirre et al., 2004). Thismeans that drugs found for CF treatment may also be effective in thetreatment of other diseases.

No therapy currently exists that restores the function of mutant CFTR.Restoring mutant CFTR function is expected to decrease CF-associatedcomplications, and improve quality of life and expected life-span of CFpatients.

Therefore, there is a clear need for molecules that facilitate thefolding, processing and/or migration of the ΔF508-CFTR to the plasmamembrane, thereby increasing the density of ΔF508-CFTR in the membrane,and rescuing the function of ΔF508-CFTR (correctors). These correctorsmay be an inhibitory agent, particularly small molecule drug compoundsor biologic drugs, which target a protein regulating the processing ofΔF508-CFTR through the ER. To enable the development of such a drug,there is a need to identify target proteins, that, when antagonized,increase the density and functional performance of ΔF508-CFTR in theplasma membrane.

An example of such a protein target is syntaxin-8 (STX8), which isinvolved in trafficking of vesicles and has been shown to bind to thewild-type CFTR (Antonin et al., 2000; Bilan et al., 2004; Thoreau etal., 1999). It has been shown that syntaxin-8 can function as a drugtarget by correcting CF-associated mutant CFTR function (Fischer et al.,2006). Another positive control is BCAP31 (Lambert et al., 2001). It hasbeen previously demonstrated that down-regulation of BCAP31 by Ad-siRNAallows functional restoration of ΔF508-CFTR (Fischer et al., 2006).

Therefore, there remains a need to identify further targets which may beof use in the diagnosis, prevention and or treatment of disordersinvolving ER-associated protein misfolding and in particular diseasescharacterized by abnormal trafficking of a disease-associated protein.Exemplary conditions include, but are not limited to, Cystic Fibrosis,Parkinson's disease, Gaucher's disease, nephrogenic diabetes insipidus,emphysema and liver disease, Maple syrup urine disease, Fabry's disease,hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia,beta-galactosidosis, Wilson's disease, long QT syndrome and retinitispimentosa, transthyretin-linked amyloidosis, Alzheimer's disease, priondisease, and inclusion body myositis. In particular the disease isCystic Fibrosis. As many of the clinical symptoms (e.g. airwayobstruction, chronic inflammation, mucus overproduction, enhancedcytokine production) of CF overlap with those of asthma and COPD(Chronic Obstructive Pulmonary Disease), these targets may also be ofuse in the diagnosis, prevention and or treatment of asthma and COPD.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that agents whichinhibit the expression and/or activity of the TARGETS disclosed hereinare able to result in the increased functional activity of CF-associatedmutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) inhuman airway bronchial epithelial cells. The present invention thereforeprovides TARGETS which are involved in the pathway involved in cellulartrafficking/protein trafficking and/or folding, methods for screeningfor agents capable of modulating the expression and/or activity ofTARGETS and uses of these agents in the prevention and/or treatment ofER-associated protein misfolding diseases, in particular CysticFibrosis.

The present invention relates to a method for identifying compounds thatincrease the functional activity of CF-associated mutant CFTR,comprising contacting the compound with the identified TARGETS or theirprotein domain fragments (SEQ ID. NO 30-55) under conditions that allowsaid TARGETS or their protein domain fragments to bind to the compound,and measuring a compound-polypeptide property related to the increasedfunctional activity of CF-associated mutant CFTR.

In particular the present invention provides TARGETS which are involvedin the pathway involved in cellular trafficking, particularly of CFTR,methods for screening for agents capable of modulating the expressionand/or activity of TARGETS and uses of these agents in the preventionand/or treatment of CF. The present invention provides TARGETS which areinvolved in or otherwise associated with airway epithelial cellfunction. The present invention provides TARGETS which are involved ininflammation and the inflammatory response, particularly associated withCF and/or in airway epithelial cells. The invention provides uses ofagents directed against these targets in CF and other airway diseasesinvolving an inflammatory aspect or component, including asthma andCOPD.

Aspects of the present method include the in vitro assay of compoundsusing identified TARGETS, and cellular assays wherein identified TARGETinhibition is followed by observing indicators of efficacy, includingchloride channel activity. Another aspect of the invention is a methodof treatment or prevention of a condition involving a decrease infunctional activity of CF-associated mutant CFTR, in a subject sufferingor susceptible thereto, by administering a pharmaceutical compositioncomprising an effective corrector for enhancing the functional activityof CF-associated mutant CFTR.

The present invention relates to a method for identifying compounds thatinhibit the TARGET(s), comprising contacting the compound with theidentified TARGETS (SEQ ID NO: 30-55) or their protein domain fragmentsunder conditions wherein the compounds may interact with or influencethe TARGET(s), measuring the expression or activity of a protein whichis misfolded in an ER-associated protein misfolding disease, andselecting compounds which increase the expression or activity of theprotein which is misfolded in the ER-associated protein misfoldingdisease. In one such method the expression or activity of ΔF508 CFTR,misfolded in the disease CF, is measured. In exemplary further suchmethods, the expression or activity of fibrillin, misfolded in Marfansyndrome, or of alpha galactosidase, misfolded in Fabry's disease, or ofrhodopsin, misfolded in retinitis pigmentosa, or beta-amyloid protein,misfolded in Alzheimer's disease, is/are measured, and compounds whichincrease the proper expression or activity thereof are selected.

The present invention relates to a method for identifying compounds thatare able to modulate protein folding and trafficking, and particularlyER-associated protein folding and cellular trafficking, comprisingcontacting a compound with a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 30-55(hereinafter “TARGETS”) and fragments thereof, under conditions thatallow said polypeptide to bind to said compound, and measuring acompound-polypeptide property related to cellular trafficking ofproteins. In a specific embodiment, the present invention relates to amethod for identifying compounds that are able to modulate the proteinfolding, trafficking or activity of the mutant CFTR protein in airwayepithelial cells, comprising contacting a compound with a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 30-55 (hereinafter “TARGETS”) and fragments thereof, underconditions that allow said polypeptide to bind to said compound, andmeasuring a compound-polypeptide property related to CFTR expression oractivity. In a specific embodiment the compound-polypeptide propertymeasured is CFTR-mediated halide flux. In a specific embodiment, theproperty measured is CFTR expression on the cell surface.

Aspects of the present method include the in vitro assay of compoundsusing polypeptide of a TARGET, or fragments thereof, such fragmentsincluding the amino acid sequences described by SEQ ID NO: 30-55 andcellular assays wherein TARGET inhibition is followed by observingindicators of efficacy including, for example, TARGET expression levels,TARGET enzymatic activity, CFTR protein levels, CFTR activity,CFTR-mediated halide flux, and/or other assessments of proteinfolding/trafficking or inflammation and inflammatory response.

The present invention also relates to

-   -   (1) expression inhibitory agents comprising a polynucleotide        selected from the group of an antisense polynucleotide, a        ribozyme, and a small interfering RNA (siRNA), wherein said        polynucleotide comprises a nucleic acid sequence complementary        to, or engineered from, a naturally occurring polynucleotide        sequence encoding a TARGET polypeptide said polynucleotide        sequence comprising a sequence selected from the group        consisting of SEQ ID NO: 1-29 and    -   (2) pharmaceutical compositions comprising said agent(s), useful        in the treatment, or prevention, of a disease characterized by        ER-associated protein misfolding, including in particular Cystic        Fibrosis.

Another aspect of the invention is a method of treatment, or preventionof a condition related to a disease characterized by ER-associatedprotein misfolding, in particular Cystic Fibrosis, in a subjectsuffering or susceptible thereto, by administering a pharmaceuticalcomposition comprising an effective TARGET-expression inhibiting amountof a expression-inhibitory agent or an effective TARGET activityinhibiting amount of a activity-inhibitory agent.

A further aspect of the present invention is a method for diagnosis of adisease characterized by ER-associated protein misfolding comprisingmeasurement of indicators of levels of TARGET expression in a subject.In particular the present invention relates to a method for thediagnosis of Cystic Fibrosis.

Another aspect of this invention relates to the use of agents whichinhibit a TARGET as disclosed herein in a therapeutic method, apharmaceutical composition, and the manufacture of such composition,useful for the treatment of a disease involving protein misfolding. Inparticular, the present method relates to the use of the agents whichinhibit a TARGET in the treatment of a disease characterized byER-associated protein misfolding, and in particular, a diseasecharacterized by abnormal trafficking of a disease-associated protein.Suitable conditions include, but are not limited to, Cystic Fibrosis,Parkinson's disease, Gaucher's disease, nephrogenic diabetes insipidus,emphysema and liver disease (alpha-1-antitrypsin deficiency), Maplesyrup urine disease, Fabry's disease, hypogonadotropic hypogonadism,hyperinsulinemic hypoglycemia, beta-galactosidosis, Wilson's disease,long QT syndrome, retinitis pigmentosa, transthyretin-linkedamyloidosis, Alzheimer's disease, prion disease, and inclusion bodymyositis. In particular the disease is Cystic Fibrosis.

Other objects and advantages will become apparent from a considerationof the ensuing description taken in conjunction with the followingillustrative drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: Expression of halide-sensitive fluorescent protein YFP

FIG. 2: YFP halide transport in CFBE cells transduced with ΔF508 CFTRvirus and wild type CFTR cells

FIG. 3: Example of a control plate during Ad-siRNA screening

FIG. 4: High-throughput screening data on 11,330 Ad-siRNAs in theCFTR-Dependent Halide Flux Assay.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

The term ‘agent’ means any molecule, including polypeptides, antibodies,polynucleotides, chemical compounds and small molecules. In particularthe term agent includes compounds such as test compounds or drugcandidate compounds.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense.

The term ‘assay’ means any process used to measure a specific propertyof a compound. A ‘screening assay’ means a process used to characterizeor select compounds based upon their activity from a collection ofcompounds.

The term ‘binding affinity’ is a property that describes how stronglytwo or more compounds associate with each other in a non-covalentrelationship. Binding affinities can be characterized qualitatively,(such as ‘strong’, ‘weak’, ‘high’, or ‘low’) or quantitatively (such asmeasuring the K_(D)).

The term ‘carrier’ means a non-toxic material used in the formulation ofpharmaceutical compositions to provide a medium, bulk and/or useableform to a pharmaceutical composition. A carrier may comprise one or moreof such materials such as an excipient, stabilizer, or an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude aqueous or solid buffer ingredients including phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

The term ‘complex’ means the entity created when two or more compoundsbind to, contact, or associate with each other.

The term ‘compound’ is used herein in the context of a ‘test compound’or a ‘drug candidate compound’ described in connection with the assaysof the present invention. As such, these compounds comprise organic orinorganic compounds, derived synthetically, recombinantly, or fromnatural sources.

The compounds include inorganic or organic compounds such aspolynucleotides, lipids or hormone analogs. Other biopolymeric organictest compounds include peptides comprising from about 2 to about 40amino acids and larger polypeptides comprising from about 40 to about500 amino acids, including polypeptide ligands, enzymes, receptors,channels, antibodies or antibody conjugates.

The term ‘condition’ or ‘disease’ means the overt presentation ofsymptoms (i.e., illness) or the manifestation of abnormal clinicalindicators (for example, biochemical indicators or diagnosticindicators). Alternatively, the term ‘disease’ refers to a genetic orenvironmental risk of or propensity for developing such symptoms orabnormal clinical indicators.

The term ‘contact’ or ‘contacting’ means bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

The term ‘derivatives of a polypeptide’ relates to those peptides,oligopeptides, polypeptides, proteins and enzymes that comprise astretch of contiguous amino acid residues of the polypeptide and thatretain a biological activity of the protein, for example, polypeptidesthat have amino acid mutations compared to the amino acid sequence of anaturally-occurring form of the polypeptide. A derivative may furthercomprise additional naturally occurring, altered, glycosylated, acylatedor non-naturally occurring amino acid residues compared to the aminoacid sequence of a naturally occurring form of the polypeptide. It mayalso contain one or more non-amino acid substituents, or heterologousamino acid substituents, compared to the amino acid sequence of anaturally occurring form of the polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence.

The term ‘derivatives of a polynucleotide’ relates to DNA-molecules,RNA-molecules, and oligonucleotides that comprise a stretch of nucleicacid residues of the polynucleotide, for example, polynucleotides thatmay have nucleic acid mutations as compared to the nucleic acid sequenceof a naturally occurring form of the polynucleotide. A derivative mayfurther comprise nucleic acids with modified backbones such as PNA,polysiloxane, and 2′-O-(2-methoxy)ethyl-phosphorothioate, non-naturallyoccurring nucleic acid residues, or one or more nucleic acidsubstituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleosides, or a reportermolecule to facilitate its detection.

The term ‘endogenous’ shall mean a material that a cell or mammal (asthe context dictates) naturally produces. Endogenous in reference to theterm ‘protease’, ‘kinase’, or G-Protein Coupled Receptor (‘GPCR’) shallmean that which is naturally produced by a cell, for example a mammaliancell (for example, not limitation a human cell), or by a mammal (forexample, and not limitation, a human). In contrast, the termnon-endogenous in this context shall mean that which is not naturallyproduced by a cell, in particular by a mammalian cell, (for example, notlimitation a human cell), or that is not naturally produced by a mammal(for example, and not limitation, a human). Both terms can be utilizedto describe both in vivo and in vitro systems. For example, and withoutlimitation, in a screening approach, the endogenous or non-endogenousTARGET may be in reference to an in vitro screening system. As a furtherexample and not limitation, where the genome of a mammal has beenmanipulated to include a non-endogenous TARGET, screening of a candidatecompound by means of an in vivo system is viable.

The term ‘expressible nucleic acid’ means a nucleic acid coding for aproteinaceous molecule, an RNA molecule, or a DNA molecule.

The term ‘expression’ comprises both endogenous expression andoverexpression by transduction.

The term ‘expression inhibitory agent’ means a polynucleotide designedto interfere selectively with the transcription, translation and/orexpression of a specific polypeptide or protein normally expressedwithin a cell. More particularly, ‘expression inhibitory agent’comprises a DNA or RNA molecule that contains a nucleotide sequenceidentical to or complementary to at least about 15-30, particularly atleast 17, sequential nucleotides within the polyribonucleotide sequencecoding for a specific polypeptide or protein. Exemplary expressioninhibitory molecules include ribozymes, double stranded siRNA molecules,self-complementary single-stranded siRNA molecules, genetic antisenseconstructs, and synthetic RNA antisense molecules with modifiedstabilized backbones.

The term ‘fragment of a polynucleotide’ relates to oligonucleotides thatcomprise a stretch of contiguous nucleic acid residues that exhibitsubstantially a similar, but not necessarily identical, activity as thecomplete sequence. In a particular aspect, ‘fragment’ may refer to aoligonucleotide comprising a nucleic acid sequence of at least 5 nucleicacid residues (preferably, at least 10 nucleic acid residues, at least15 nucleic acid residues, at least 20 nucleic acid residues, at least 25nucleic acid residues, at least 40 nucleic acid residues, at least 50nucleic acid residues, at least 60 nucleic residues, at least 70 nucleicacid residues, at least 80 nucleic acid residues, at least 90 nucleicacid residues, at least 100 nucleic acid residues, at least 125 nucleicacid residues, at least 150 nucleic acid residues, at least 175 nucleicacid residues, at least 200 nucleic acid residues, or at least 250nucleic acid residues) of the nucleic acid sequence of said completesequence.

The term ‘fragment of a polypeptide’ relates to peptides, oligopeptides,polypeptides, proteins, monomers, subunits and enzymes that comprise astretch of contiguous amino acid residues, and exhibit substantially asimilar, but not necessarily identical, functional or expressionactivity as the complete sequence. In a particular aspect, ‘fragment’may refer to a peptide or polypeptide comprising an amino acid sequenceof at least 5 amino acid residues (preferably, at least 10 amino acidresidues, at least 15 amino acid residues, at least 20 amino acidresidues, at least 25 amino acid residues, at least 40 amino acidresidues, at least 50 amino acid residues, at least 60 amino residues,at least 70 amino acid residues, at least 80 amino acid residues, atleast 90 amino acid residues, at least 100 amino acid residues, at least125 amino acid residues, at least 150 amino acid residues, at least 175amino acid residues, at least 200 amino acid residues, or at least 250amino acid residues) of the amino acid sequence of said completesequence.

The term ‘hybridization’ means any process by which a strand of nucleicacid binds with a complementary strand through base pairing. The term‘hybridization complex’ refers to a complex formed between two nucleicacid sequences by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(for example, C_(0t) or R_(0t) analysis) or formed between one nucleicacid sequence present in solution and another nucleic acid sequenceimmobilized on a solid support (for example, paper, membranes, filters,chips, pins or glass slides, or any other appropriate substrate to whichcells or their nucleic acids have been fixed). The term “stringentconditions” refers to conditions that permit hybridization betweenpolynucleotides and the claimed polynucleotides. Stringent conditionscan be defined by salt concentration, the concentration of organicsolvent, for example, formamide, temperature, and other conditions wellknown in the art. In particular, reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature can increase stringency. The term ‘standard hybridizationconditions’ refers to salt and temperature conditions substantiallyequivalent to 5×SSC and 65° C. for both hybridization and wash. However,one skilled in the art will appreciate that such ‘standard hybridizationconditions’ are dependent on particular conditions including theconcentration of sodium and magnesium in the buffer, nucleotide sequencelength and concentration, percent mismatch, percent formamide, and thelike. Also important in the determination of “standard hybridizationconditions” is whether the two sequences hybridizing are RNA-RNA,DNA-DNA or RNA-DNA. Such standard hybridization conditions are easilydetermined by one skilled in the art according to well known formulae,wherein hybridization is typically 10-20^(N)C below the predicted ordetermined T_(m) with washes of higher stringency, if desired.

The term ‘inhibit’ or ‘inhibiting’, in relationship to the term‘response’ means that a response is decreased or prevented in thepresence of a compound as opposed to in the absence of the compound.

The term ‘inhibition’ refers to the reduction, down regulation of aprocess or the elimination of a stimulus for a process, which results inthe absence or minimization of the expression or activity of a proteinor polypeptide.

The term ‘induction’ refers to the inducing, up-regulation, orstimulation of a process, which results in the expression or activity ofa protein or polypeptide.

The term ‘ligand’ means an endogenous, naturally occurring moleculespecific for an endogenous, naturally occurring receptor.

The term ‘pharmaceutically acceptable salts’ refers to the non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds which inhibit the expression or activity of TARGETS asdisclosed herein. These salts can be prepared in situ during the finalisolation and purification of compounds useful in the present invention.

The term ‘polypeptide’ relates to proteins (such as TARGETS),proteinaceous molecules, fragments of proteins, monomers, subunits orportions of polymeric proteins, peptides, oligopeptides and enzymes(such as kinases, proteases, GPCR's etc.).

The term ‘polynucleotide’ means a polynucleic acid, in single or doublestranded form, and in the sense or antisense orientation, complementarypolynucleic acids that hybridize to a particular polynucleic acid understringent conditions, and polynucleotides that are homologous in atleast about 60 percent of its base pairs, and more particularly 70percent of its base pairs are in common, most particularly 90 percent,and in a particular embodiment, 100 percent of its base pairs. Thepolynucleotides include polyribonucleic acids, polydeoxyribonucleicacids, and synthetic analogues thereof. It also includes nucleic acidswith modified backbones such as peptide nucleic acid (PNA),polysiloxane, and 2′-O-(2-methoxy)ethylphosphorothioate. Thepolynucleotides are described by sequences that vary in length, thatrange from about 10 to about 5000 bases, particularly about 100 to about4000 bases, more particularly about 250 to about 2500 bases. Onepolynucleotide embodiment comprises from about 10 to about 30 bases inlength. A particular embodiment of polynucleotide is thepolyribonucleotide of from about 17 to about 22 nucleotides, morecommonly described as small interfering RNAs (siRNAs). Anotherparticular embodiment are nucleic acids with modified backbones such aspeptide nucleic acid (PNA), polysiloxane, and2′-O-(2-methoxy)ethylphosphorothioate, or including non-naturallyoccurring nucleic acid residues, or one or more nucleic acidsubstituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleosides, or a reportermolecule to facilitate its detection. Polynucleotides herein areselected to be ‘substantially’ complementary to different strands of aparticular target DNA sequence. This means that the polynucleotides mustbe sufficiently complementary to hybridize with their respectivestrands. Therefore, the polynucleotide sequence need not reflect theexact sequence of the target sequence. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the polynucleotide,with the remainder of the polynucleotide sequence being complementary tothe strand. Alternatively, non-complementary bases or longer sequencescan be interspersed into the polynucleotide, provided that thepolynucleotide sequence has sufficient complementarity with the sequenceof the strand to hybridize therewith under stringent conditions or toform the template for the synthesis of an extension product.

The term ‘preventing’ or ‘prevention’ refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term ‘prophylaxis’ is related to and encompassed in the term‘prevention’, and refers to a measure or procedure the purpose of whichis to prevent, rather than to treat or cure a disease. Non-limitingexamples of prophylactic measures may include the administration ofvaccines; the administration of low molecular weight heparin to hospitalpatients at risk for thrombosis due, for example, to immobilization; andthe administration of an anti-malarial agent such as chloroquine, inadvance of a visit to a geographical region where malaria is endemic orthe risk of contracting malaria is high.

The term ‘solvate’ means a physical association of a compound useful inthis invention with one or more solvent molecules. This physicalassociation includes hydrogen bonding. In certain instances the solvatewill be capable of isolation, for example when one or more solventmolecules are incorporated in the crystal lattice of the crystallinesolid. ‘Solvate’ encompasses both solution-phase and isolable solvates.Representative solvates include hydrates, ethanolates and methanolates.

The term ‘subject’ includes humans and other mammals.

‘Therapeutically effective amount’ means that amount of a drug,compound, expression inhibitory agent, or pharmaceutical agent that willelicit the biological or medical response of a subject that is beingsought by a medical doctor or other clinician. In particular, withregard to increasing the functional activity of CF-associated mutantCFTR, the term “effective amount” is intended to include an effectiveamount of a compound or agent that will bring about a biologicallymeaningful increase in CFTR-dependent halide flux.

The term ‘treating’ or ‘treatment’ of any disease or disorder refers, inone embodiment, to ameliorating the disease or disorder (i.e., arrestingthe disease or reducing the manifestation, extent or severity of atleast one of the clinical symptoms thereof). In another embodiment‘treating’ or ‘treatment’ refers to ameliorating at least one physicalparameter, which may not be discernible by the subject. In yet anotherembodiment, ‘treating’ or ‘treatment’ refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In a further embodiment, ‘treating’ or ‘treatment’relates to slowing the progression of the disease.

The term ‘vectors’ also relates to plasmids as well as to viral vectors,such as recombinant viruses, or the nucleic acid encoding therecombinant virus.

The term ‘vertebrate cells’ means cells derived from animals havingvertebral structure, including fish, avian, reptilian, amphibian,marsupial, and mammalian species. Preferred cells are derived frommammalian species, and most preferred cells are human cells. Mammaliancells include feline, canine, bovine, equine, caprine, ovine, porcineand murine, such as mice and rats, and rabbits.

The term ‘TARGET’ or ‘TARGETS’ means the protein(s) identified inaccordance with the assays described herein and determined to beinvolved in the modulation of a Cystic Fibrosis phenotype. The termTARGET or TARGETS includes and contemplates alternative species forms,isoforms, and variants, such as allelic variants, including as a resultof allelic or natural variation in the amino acid sequence, and splicevariants, alternate in frame exons, and alternative or prematuretermination or start sites, including known or recognized isoforms orvariants thereof such as indicated in Table 1.

The term ‘disease characterized by ER-associated protein misfolding’refers to a disease which involves, results at least in part from, orincludes a protein misfolding aspect, particularly wherein a protein isnot processed and/or sorted by or through the endoplasmic reticulum (ER)in a proper, efficient, or effective manner, such that it ismisprocessed, poorly processed, degraded, or misfolded, resulting insuch instances in less protein processed to the cell membrane or otherprotein location destination, or in processed protein having reduced oraltered activity. The term includes, but is not limited to, exemplarydiseases selected from Cystic Fibrosis, Parkinson's disease, Gaucher'sdisease, nephrogenic diabetes insipidus, emphysema and liver disease(alpha-1 antitrypsin deficiency), Maple syrup urine disease, Fabry'sdisease, hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia,beta-galactosidosis, Wilson's disease, long QT syndrome retinitispigmentosa, transthyretin-linked amyloidosis, Alzheimer's disease, priondisease, and inclusion body myositis. Such diseases can be associatedwith misfolding of proteins, or alternatively folded proteins, includingmisfolded CFTR (Cystic Fibrosis), misfolded fibrillin (Marfan syndrome),misfolded alpha gatactosidase (Fabry's disease), misfolded betaglucocerebrosidase (Gaucher's disease), misfolded hERG receptor (long QTsyndrome), misfolded rhodopsin (retinitis pigmentosa), misfolded oralternatively folded beta amyloid protein (Alzheimer's disease), andmisfolded or alternatively folded prion protein (Prion Disease).

Targets

The present invention is based on the present inventors' discovery thatthe TARGETS are factors in the translocation of ΔF508 CFTR to the plasmamembrane, whereby inhibition of the TARGETS results in an increase inCFTR-mediated halide flux. The TARGETS are factors or protein moleculesinvolved in protein trafficking and/or folding such that theirinhibition results in an increased amount of ΔF508 CFTR being traffickedto, expressed, and/or active at the plasma membrane. The TARGETS mayalso serve a role in inflammation and/or the inflammatory response,particularly in pulmonary epithelial cells. In the present application,the effect of down-regulation of syntaxin-8, which is involved intrafficking of vesicles and has been shown to bind to the wild type CFTR(Antonin et al. 2000; Bilan et al., 2004; Thoreau et al., 1999), orBCAP31, for which down-regulation by Ad-siRNA allows functionalrestoration of ΔF508 CFTR (Fischer et al., 2006), is used as a positivecontrol in a screen of 11,330 Ad-siRNAs to identify novel TARGETS.

CFTR is an ion channel. Ion channels are membrane protein complexes andtheir function is to facilitate the diffusion of ions across biologicalmembranes. Membranes, or phospholipid bilayers, build a hydrophobic, lowdielectric barrier to hydrophilic and charged molecules. Ion channelsprovide a high conducting, hydrophilic pathway across the hydrophobicinterior of the membrane. The activity of an ion channel can be measuredusing classical patch clamping. High-throughput fluorescence-based ortracer-based assays are also widely available to measure ion channelactivity. These fluorescent-based assays screen compounds on the basisof their ability to either open or close an ion channel thereby changingthe concentration of specific fluorescent dyes across a membrane. In thecase of the tracer based assay, the changes in concentration of thetracer within and outside the cell are measured by radioactivitymeasurement or gas absorption spectrometry.

The TARGETS listed in Table 1 below were identified herein as involvedin the modulation of the migration of ΔF508-CFTR to the plasma membrane,therefore, inhibitors of these TARGETS are able to increase the densityof ΔF508-CFTR in the membrane, and rescue the function of ΔF508-CFTR.These TARGETS are proposed to have a general role in modulating thefolding of proteins within the ER and their subsequent trafficking tothe cell membrane. Therefore these TARGETS are involved in diseasescharacterized by ER-associated protein misfolding, in particular CysticFibrosis.

Therefore, in one aspect, the present invention relates to a method forassaying for drug candidate compounds that modulate trafficking of adisease-associated protein comprising contacting the compound with apolypeptide comprising an amino acid sequence of SEQ ID NO: 30-55, orfragment thereof, under conditions that allow said polypeptide to bindto the compound, and detecting the formation of a complex between thepolypeptide and the compound. In particular said method is used toidentify an agent that increases the functional activity ofCF-associated mutant CFTR said method. In particular said method may beused to identify drug candidate compounds that promote migration ofΔF508-CFTR to the plasma membrane. One particular means of measuring thecomplex formation is to determine the binding affinity of said compoundto said polypeptide.

More particularly, the invention relates to a method for identifying anagent or compound that increases the functional activity ofCF-associated mutant CFTR said method comprising:

-   -   (a) contacting a population of mammalian cells with one or more        compound that exhibits binding affinity for a TARGET        polypeptide, or fragment thereof, and    -   (b) measuring a compound-polypeptide property related to        ΔF508-CFTR activity or expression.

In a further aspect of the present invention said method is used toidentify a compound that increases the activity or expression ofCF-associated mutant CFTR by promoting migration or trafficking ofΔF508-CFTR to the plasma membrane.

In a further aspect, the present invention relates to a method forassaying for drug candidate compounds that modulate trafficking of adisease-associated protein comprising contacting the compound with apolypeptide comprising an amino acid sequence of SEQ ID NO: 30-55, orfragment thereof, under conditions that allow said compound to modulatethe activity or expression of the polypeptide, and determining theactivity or expression of the polypeptide. In particular said method maybe used to identify drug candidate compounds capable of promoting themigration of ΔF508-CFTR to the plasma membrane. One particular means ofmeasuring the activity or expression of the polypeptide is to determinethe amount of said polypeptide using a polypeptide binding agent, suchas an antibody, or to determine the activity of said polypeptide in abiological or biochemical measure, for instance the amount ofphosphorylation of a target of a kinase polypeptide.

The compound-polypeptide property referred to above is related to theexpression and/or activity of the TARGET, and is a measurable phenomenonchosen by the person of ordinary skill in the art. The measurableproperty may be, for example, the binding affinity of said compound fora peptide domain of the polypeptide TARGET, a property related to thefolding or activity of the disease-related protein or the level of anyone of a number of biochemical marker levels of CF-associated mutantCFTR activity. In a preferred method, CF-associated mutant CFTR activityis measured by measuring CFTR-dependent halide flux, which can bemonitored by using a reporter protein, halide-sensitive fluorescentprotein YFP. It has been reported that cells expressing this reporterprotein show enhanced fluorescence quenching of YFP by extracellularisomolar iodide solutions in the presence of activated CFTR (Galietta etal., 2001b). Fluorescence quenching is a measure of halide transport—ashalide ions cross the plasma membrane, the halide ions interact withhalide-sensitive fluorescent protein YFP, and quench the fluorescence ofYFP. Fluorescence quenching is measured on a fluorescence plate reader.

In an additional aspect, the present invention relates to a method forassaying for drug candidate compounds that modulate trafficking of adisease-associated protein, comprising contacting the compound with anucleic acid encoding a TARGET polypeptide, including a nucleic acidsequence selected from SEQ ID NO: 1-29, or fragment/portion thereof,under conditions that allow said nucleic acid to bind to or otherwiseassociate with the compound, and detecting the formation of a complexbetween the nucleic acid and the compound. In particular, said methodmay be used to identify drug candidate compounds able to promotemigration of ΔF508-CFTR to the plasma membrane. One particular means ofmeasuring the complex formation is to determine the binding affinity ofsaid compound to said nucleic acid or the presence of a complex byvirtue of resistance to nucleases or by gel mobility assays.Alternatively, complex formation may be determined by inhibition ofnucleic acid transcription or translation.

In a particular embodiment of the invention, the TARGET polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID No: 30-55 as listed in Table 1. In an embodiment of theinvention, the nucleic acid capable of encoding the TARGET polypeptidecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1-29 as listed in Table 1. Table 1 provides TARGET exemplaryhuman nucleic acid and protein sequence, including recognized variantsor isoforms where more than one accession number and SEQ ID NO: isindicated. Isoforms or variants of the TARGET(S) include nucleic acid orproteins with or utilizing alternate in frame exons, alternativesplicing or splice variants, and alternative or premature terminationvariants.

TABLE 1 TARGETS TARGET SEQ ID SEQ ID Gene GenBank nucleic NO: GenBankNO: Symbol acid Acc#: DNA protein Acc#: Protein Class UGT3A1 NM_152404 1NP_689617 30 enzyme UGT3A2 NM_174914 2 NP_777574 31 enzyme PHGDHNM_006623 3 NP_006614 32 enzyme B3GNT3 NM_014256 4 NP_055071 33 enzymePPIH NM_006347 5 NP_006338 34 enzyme/chaperone CELSR3 NM_001407 6NP_001398 35 GPCR MC2R NM_000529 7 NP_000520 36 GPCR MAS1L NM_052967 8NP_443199 37 GPCR LRRK2 NM_198578 9 NP_940980 38 kinase/nucleotidebinding NLRP1 NM_001033053 10 NP_001028225 39 kinase/nucleotideNM_014922 11 NP_055737 40 binding NM_033004 12 NP_127497 41 NM_033006 13NP_127499 42 NM_033007 14 PMS1 NM_000534 15 NP_000525 43kinase/nucleotide NM_001128143 16 binding NM_001128144 17 MAK NM_00590618 NP_005897 44 kinase/nucleotide binding CPD NM_001304 19 NP_001295 45peptidase/peptidase inhibitor CST7 NM_003650 20 NP_003641 46peptidase/peptidase inhibitor DUSP5 NM_004419 21 NP_004410 47phosphatase PTPRG NM_002841 22 NP_002832 48 phosphatase/receptor IL6RNM_000565 23 NP_000556 49 receptor NM_181359 24 NP_852004 50 GHRNM_000163 25 NP_000154 51 receptor CSF3 NM_000759 26 NP_000750 52secreted NM_172219 27 NP_757373 53 NM_172220 28 NP_757374 54 SPNS1NM_032038 29 NP_114427 55 transporter

Depending on the choice of the skilled artisan, the present assay methodmay be designed to function as a series of measurements, each of whichis designed to determine whether the drug candidate compound is indeedacting on the TARGET to thereby increase the functional activity ofCF-associated mutant CFTR. For example, an assay designed to determinethe binding affinity of a compound to the TARGET, or fragment thereof,may be necessary, but not sufficient, to ascertain whether the testcompound would be useful for increasing the functional activity ofCF-associated mutant CFTR when administered to a subject. Nonetheless,such binding information would be useful in identifying a set of testcompounds for use in an assay that would measure a different property,further down the biochemical pathway, such as halide flux, assayed bymeasuring the quenching of a halide-sensitive fluorescent protein. Suchadditional assay(s) may be designed to confirm that the test compound,having binding affinity for the TARGET, actually increases thefunctional activity of CF-associated mutant CFTR.

Suitable controls should always be in place to insure against falsepositive readings. In a particular embodiment of the present inventionthe screening method comprises the additional step of comparing thecompound to a suitable control. In one embodiment, the control may be acell or a sample that has not been in contact with the test compound. Inan alternative embodiment, the control may be a cell that does notexpress the TARGET; for example in one aspect of such an embodiment thetest cell may naturally express the TARGET and the control cell may havebeen contacted with an agent, e.g. an siRNA, which inhibits or preventsexpression of the TARGET. Alternatively, in another aspect of such anembodiment, the cell in its native state does not express the TARGET andthe test cell has been engineered so as to express the TARGET, so thatin this embodiment, the control could be the untransformed native cell.Whilst exemplary controls are described herein, this should not be takenas limiting; it is within the scope of a person of skill in the art toselect appropriate controls for the experimental conditions being used.

The order of taking these measurements is not believed to be critical tothe practice of the present invention, which may be practiced in anyorder. For example, one may first perform a screening assay of a set ofcompounds for which no information is known respecting the compounds'binding affinity for the TARGET. Alternatively, one may screen a set ofcompounds identified as having binding affinity for a TARGET proteindomain, or a class of compounds identified as being an inhibitor of theTARGET. However, for the present assay to be meaningful to the ultimateuse of the drug candidate compounds in diseases characterized byER-associated protein misfolding a measurement of functional activity orappropriate expression of the relevant protein is necessary. In aspecific embodiment the disease is cystic fibrosis and the protein isCF-associated mutant CFTR. In alternative embodiments, the disease isMarfan syndrome and the protein is fibrillin, or the disease is Fabry'sdisease and the protein is alpha gatactosidase, or the disease isGaucher's disease and the protein is beta glucocerebrosidase, or thedisease is long QT syndrome and the protein is misfolded hERG receptor,or the disease is retinitis pigmentosa and the protein is rhodopsin, orthe disease is Alzheimer's disease and the protein is beta-amyloid orthe disease is prion disease and the protein is prion protein.Validation studies, including controls, and measurements of bindingaffinity to the polypeptides of the invention are nonetheless useful inidentifying a compound useful in any therapeutic or diagnosticapplication.

Analogous approaches based on art-recognized methods and assays may beapplicable with respect to the TARGETS and compounds in any of variousdisease(s) characterized by ER-associated protein misfolding orinflammatory diseases, including airway epithelial cell diseases,asthma, COPD. An assay or assays may be designed to confirm that thetest compound, having binding affinity for the TARGET, increases thefunctional activity and/or alters the protein misfolding or proteintrafficking of a protein associated with misfolding disease. In one suchmethod the expression or activity of ΔF508 CFTR, misfolded in thedisease CF, is measured. In the case of CF, and in lieu of animalmodels, chambers with primary human airway epithelial cells (Li et al,2004) may be utilized in further assessing the TARGETS and/or compounds.In exemplary further such methods, the expression or activity offibrillin, misfolded in Marfan syndrome, or of alpha galactosidase,misfolded in Fabry disease, or of rhodopsin, misfolded ion retinitispigmentosa, or beta amyloid protein, misfolded in Alzheimer's disease,is/are measured, and compounds which increase the proper expression oractivity thereof are selected. Protein trafficking may be assessed ormonitored in art-recognized methods, including in vitro, ex vivo, andanimal systems.

The present assay method may be practiced in vitro, using one or more ofthe TARGET proteins, or fragments thereof, including monomers, portionsor subunits of polymeric proteins, peptides, oligopeptides andenzymatically active portions thereof.

The binding affinity of the compound with the TARGET or a fragmentthereof can be measured by methods known in the art, such as usingsurface plasmon resonance biosensors (Biacore), by saturation bindinganalysis with a labeled compound (e.g. Scatchard and Lindmo analysis),by differential UV spectrophotometer, fluorescence polarization assay,Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescenceresonance energy transfer, and Bioluminescence resonance energytransfer. The binding affinity of compounds can also be expressed indissociation constant (Kd) or as IC₅₀ or EC₅₀. The IC₅₀ represents theconcentration of a compound that is required for 50% inhibition ofbinding of another ligand to the polypeptide. The EC₅₀ represents theconcentration required for obtaining 50% of the maximum effect in anyassay that measures the TARGET function. The dissociation constant, Kd,is a measure of how well a ligand binds to the polypeptide, it isequivalent to the ligand concentration required to saturate exactly halfof the binding-sites on the polypeptide. Compounds with a high affinitybinding have low Kd, IC₅₀ and EC₅₀ values, i.e. in the range of 100 nMto 1 pM; a moderate to low affinity binding relates to a high Kd, IC₅₀and EC₅₀ values, i.e. in the micromolar range.

The present assay method may also be practiced in a cellular assay. Ahost cell expressing the TARGET can be a cell with endogenous expressionor a cell over-expressing the TARGET e.g. by transduction. When theendogenous expression of the polypeptide is not sufficient to determinea baseline that can easily be measured, one may use host cells thatover-express the TARGET. Over-expression has the advantage that thelevel of the TARGET substrate end products is higher than the activitylevel by endogenous expression. Accordingly, measuring such levels usingpresently available techniques is easier. In one such cellular assay,the biological activity of the TARGET may be measured by measuring thefunctional activity of for instance CF-associated mutant CFTR.

One embodiment of the present method for identifying a compound thatincreases CFTR expression and/or activity comprises culturing apopulation of mammalian cells expressing a TARGET polypeptide, or afunctional fragment or derivative thereof; determining a first level ofCFTR or ΔF508-CFTR expression at the cell membrane and/or activity ofCFTR or ΔF508-CFTR in said population of cells; eventually activatingthe population of cells; exposing said population of cells to acompound, or a mixture of compounds; determining a second level of CFTRor ΔF508-CFTR expression and/or activity in said population of cellsduring or after exposure of said population of cells to said compound,or the mixture of said compounds; and identifying the compound(s) thatinduce ΔF508-CFTR migration to the cell membrane and/or CFTR orΔF508-CFTR activity.

As noted above, promotion of disease-related protein trafficking may bedetermined by measuring the expression and/or activity of the TARGETpolypeptide and/or CFTR or ΔF508-CFTR.

The expression and/or activity of CFTR or ΔF508-CFTR can be determinedby methods known in the art such as immunohistochemistry using specificantibodies, or an activity assay as described herein.

The present inventors identified TARGET genes involved indisease-related protein trafficking by using a ‘knock-down’ library.This type of library is a screen in which siRNA molecules are transducedinto cells by recombinant adenoviruses, which siRNA molecules inhibit orrepress the expression of a specific gene as well as expression andactivity of the corresponding gene product in a cell. Each siRNA in aviral vector corresponds to a specific natural gene. By identifying asiRNA that promotes migration of ΔF508-CFTR to the cell membrane, adirect correlation can be drawn between the specific gene expression andthe pathway for rescuing mutant CFTR receptors. The TARGET genesidentified using the knock-down library (the protein expression productsthereof herein referred to as “TARGET” polypeptides) are then used inthe present inventive method for identifying compounds that can be usedto correct mutant CFTR expression and/or activity. Indeed, shRNAcompounds comprising the sequences listed in Table 2 (SEQ ID NOs: 56-99)inhibit the expression and/or activity of these TARGET genes and promotemigration of ΔF508-CFTR in cells, confirming the role of the TARGETS inthe protein-trafficking pathway.

TABLE 2 KD TARGET sequences useful in the practice  of the present expression-inhibitory agent  invention SEQ ID  TARGET NO:Gene SEQ ID Knock- Symbol NO: DNA Sequences Down UGT3A1 or  1, 2CGCACCTCAAGCCCTATGT; 56 UTG3A2 UGT3A2  2 AACATGGTCCGAGTAGAAG 57 PHGDH  3AGAGGAGCTGATAGCGGAG; 58 AATGGGAGCGGAAGAAGTT 59 B3GNT3  4CATCCTGCAGTGGGACTTC; 60 CAACATGGTCTTCTACCTG 61 PPIH  5GTACAAATGGCTGTCAGTT; 62 TTGAGAATGTTCCCACAGG; 63 ATGGAGATGGTACTGGAGT 64CELSR3  6 AGGATGCAGCTAACAACAA; 65 ACTGTGCGCGTACACATAA; 66ATGCTCCACAATTTGTGGC 67 MC2R  7 CATGGGCTATCTCAAGCCA; 68AACATGGGCTATCTCAAGC 69 MAS1L  8 CAGAACCCAAACCTGGTAT; 70GCCATATTGTCTCCCTTCT; 71 ACAGCAGCGCCAACCCTAT 72 LRRK2  9AAGGCTCGCGCTTCTTCTT; 73 CATTGAGACAAGAACAAGC 74 NLRP1 10, 11, AGATGGACTCTACCAAGCC; 75 12, 13,  ATTGGGAAGTCAACACTGG 76 14 PMS1 15, 16, CAGATGTTTCCGCAGCTGA; 77 17 CCAGACAATTACCCATGTA 78 MAK 18ACCTCCAAAGCAACAGAGT; 79 AGTTGTTCCCTGAATCAGT 80 CPD 19AAGTCCCAGGAAGGAGATT; 81 ACATTCACAGGTCTTTGTG 82 CST7 20CGAACGACATGTTCTTGTT; 83 CTTGTTCCCAGGACCTTAA 84 DUSP5 21TGACATTAGCTCCCACTTT; 85 ACTGGGATGGAGGAATCGG 86 PTPRG 22CCAGGAGTAGGAGGAAAGA; 87 CGGAGCAGCAAGACCATGT 88 IL6R 23, 24ACAGTCCGGCCGAAGACTT; 89 ACTATTCATGCTACCGGGC; 90 CAACATGGATGGTCAAGGA 91GHR 25 AGTGAGATGGGAAGCACCA; 92 ATGACATACATGAGGGTAC 93 CSF3 26, 27, TGGAAGAACTGGGAATGGC; 94 28 CTTTGCCACCACCATCTGG; 95 AAGCTCCTGTCCTCCCATC96 SPNS1 29 CCGCCATCTTCATTGAGGC; 97 ATCTTCTACTTTGCCATTC; 98ACTACATGGACCGCTTCAC 99

The present invention further relates to a method for identifying acompound that increases the functional activity of CF-associated mutantCFTR, comprising:

-   -   (a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        30-55;    -   (b) determining the binding affinity of the compound to the        polypeptide;    -   (c) contacting a population of mammalian cells expressing said        polypeptide with the compound that exhibits at least a moderate        binding affinity; and    -   (d) identifying the compound that increases the functional        activity of CF-associated mutant CFTR.

In one aspect, the assay method involves contacting a compound with apolypeptide comprising a fragment of an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 30-55. In one aspect, the assaymethod includes contacting cells expressing said polypeptide or fragmentwith the compound that exhibits a binding affinity in the micromolarrange. In an aspect, the binding affinity exhibited is at least 10micromolar. In one aspect, the binding affinity is at least 1micromolar. In one aspect, the binding affinity is at least 500nanomolar.

The assay method may be based on the particular expression or activityof the TARGET polypeptide, including but not limited to an enzymeactivity. Thus, assays for the enzyme TARGETs identified as SEQ ID NOs:30-34 may be based on enzymatic activity or enzyme expression. Assaysfor the peptidease/protease or peptidase inhibitor/protease inhibitorTARGETs identified as SEQ ID NOs: 45-46 may be based on proteaseactivity or expression. Assays for the kinase TARGETs identified as SEQID NOs: 38-44 may be based on kinase activity or expression, includingbut not limited to phosphorylation of a kinase target. Assays for thephosphatase TARGETs identified as SEQ ID NOs: 47-48 may be based onphosphatase activity or expression, including but not limited todephosphorylation of a phosphatase target. Assays for the GPCR andreceptor TARGETs identified as SEQ ID NO: 35-37, 48-51 may be based onGPCR activity or expression, including downstream mediators oractivators. Assays for the secreted TARGETs identified as SEQ ID NOs:52-54 may utilize activity or expression in soluble culture media orsecreted activity. Assays for the transporter TARGET identified as SEQID NOs: 55 may use techniques well known to those of skill in the artincluding classical patch clamping, high-throughput fluorescence basedor tracer based assays which measure the ability of a compound to openor close an ion channel thereby changing the concentration offluorescent dyes or tracers across a membrane or within a cell. Themeasurable phenomenon, activity or property may be selected or chosen bythe skilled artisan. The person of ordinary skill in the art may selectfrom any of a number of assay formats, systems or design one using hisknowledge and expertise in the art.

Table 1 lists the TARGETS identified using applicants' knock-downlibrary in the CFTR assay described below, including the class ofpolypeptides identified. TARGETS have been identified in polypeptideclasses including kinase, protease, enzyme, GPCR, phosphodiesterase andphosphatase, for instance. Specific methods to determine the activity ofa kinase by measuring the phosphorylation of a substrate by the kinase,which measurements are performed in the presence or absence of acompound, are well known in the art.

Specific methods to determine the inhibition by a compound by measuringthe cleavage of the substrate by the polypeptide, which is a protease,are well known in the art. Classically, substrates are used in which afluorescent group is linked to a quencher through a peptide sequencethat is a substrate that can be cleaved by the target protease. Cleavageof the linker separates the fluorescent group and quencher, giving riseto an increase in fluorescence.

G-protein coupled receptors (GPCR) are capable of activating an effectorprotein, resulting in changes in second messenger levels in the cell.The activity of a GPCR can be measured by measuring the activity levelof such second messengers. Two important and useful second messengers inthe cell are cyclic AMP (cAMP) and Ca²⁺. The activity levels can bemeasured by methods known to persons skilled in the art, either directlyby ELISA or radioactive technologies or by using substrates thatgenerate a fluorescent or luminescent signal when contacted with Ca²⁺ orindirectly by reporter gene analysis. The activity level of the one ormore secondary messengers may typically be determined with a reportergene controlled by a promoter, wherein the promoter is responsive to thesecond messenger. Promoters known and used in the art for such purposesare the cyclic-AMP responsive promoter that is responsive for thecyclic-AMP levels in the cell, and the NF-AT responsive promoter that issensitive to cytoplasmic Ca²⁺-levels in the cell. The reporter genetypically has a gene product that is easily detectable. The reportergene can either be stably infected or transiently transfected in thehost cell. Useful reporter genes are alkaline phosphatase, enhancedgreen fluorescent protein, destabilized green fluorescent protein,luciferase and β-galactosidase.

It should be understood that the cells expressing the polypeptides, maybe cells naturally expressing the polypeptides, or the cells may betransfected to express the polypeptides, as described above. Also, thecells may be transduced to overexpress the polypeptide, or may betransfected to express a non-endogenous form of the polypeptide, whichcan be differentially assayed or assessed.

In one particular embodiment the methods of the present inventionfurther comprise the step of contacting the population of cells with anagonist of the polypeptide. This is useful in methods wherein theexpression of the polypeptide in a certain chosen population of cells istoo low for a proper detection of its activity. By using an agonist thepolypeptide may be triggered, enabling a proper read-out if the compoundinhibits the polypeptide. Similar considerations apply to themeasurement of the activity of CFTR. In a particular embodiment, thecells used in the present method are mammalian lung epithelial cells.The lung epithelial cells, in the assay contemplated, may be activated(e.g. by cytokines).

A method for identifying a compound that modulates trafficking of adisease-associated protein, comprising:

-   -   (a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        30-55, and fragments thereof; and    -   (b) measuring a compound-polypeptide property related to protein        trafficking.

In one embodiment of the present invention the method relates toidentifying a compound that promotes migration of ΔF508-CFTR to theplasma membrane.

In one embodiment of the present invention the compound-polypeptideproperty related to protein trafficking is binding affinity.

In one embodiment of the present invention the compound-polypeptideproperty related to protein trafficking is increased activity ofΔF508-CFTR or CFTR.

In one embodiment of the present invention the compound-polypeptideproperty related to protein trafficking is the activity of saidpolypeptide. In particular, in one embodiment the compound inhibits theactivity of said polypeptide.

In one embodiment of the present invention the compound-polypeptideproperty related to protein trafficking is the expression of saidpolypeptide. In particular, in one embodiment the compound inhibits theexpression of said polypeptide.

The present invention further relates to a method for identifying acompound that modulates trafficking of a protein misfoldingdisease-related protein, wherein said compound exhibits at least amoderate binding affinity to an amino acid selected from the group ofSEQ ID NOS: 30-55, said method comprising:

-   -   a) contacting a compound with a population of mammalian cells        expressing a polypeptide comprising an amino acid sequence        selected from the group consisting of SEQ ID NOS: 30-55, wherein        the cells also express the protein misfolding disease-related        protein;    -   b) determining the activity or expression of the protein        misfolding disease-related protein; and    -   d) identifying the compound that modulates protein trafficking        as the compound which alters the activity or expression of the        protein misfolding disease-related protein.

In one such method, the compound exhibits a binding affinity to an aminoacid selected from the group of SEQ ID NOS: 30-55 of at least 10micromolar. In one aspect, the binding affinity is at least 1micromolar. In one aspect, the binding affinity is at least 500nanomolar.

The present invention further relates to a method for identifying acompound that modulates trafficking of a disease-related protein, saidmethod comprising:

-   -   a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        30-55;    -   b) determining the binding affinity of the compound to the        polypeptide;    -   c) contacting a population of mammalian cells expressing said        polypeptide with the compound that exhibits a binding affinity        of at least 10 micromolar; and    -   d) identifying the compound that modulates protein trafficking.

The present invention further relates to a method for identifying acompound that modulates trafficking of a disease-related protein saidmethod comprising:

-   -   a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        30-55;    -   b) determining the ability of the compound inhibit the        expression or activity of the polypeptide;    -   c) contacting a population of mammalian cells expressing said        polypeptide with the compound that significantly inhibits the        expression or activity of the polypeptide; and    -   d) identifying the compound that modulates protein trafficking.

In a particular aspect of the present invention the methods describedabove include the additional step of comparing the compound to be testedto a control, where the control is a population of cells that have notbeen contacted with the test compound.

In a particular aspect of the present invention the methods describedabove include the additional step of comparing the compound to be testedto a control, where the control is a population of cells that do notexpress said polypeptide.

For high-throughput purposes, libraries of compounds may be used such asantibody fragment libraries, peptide phage display libraries, peptidelibraries (e.g. LOPAP™, Sigma Aldrich), lipid libraries (BioMol),synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich, BioFocus DPI)or natural compound libraries (Specs, TimTec, BioFocus DPI).

Preferred drug candidate compounds are low molecular weight compounds.Low molecular weight compounds, i.e. with a molecular weight of 500Dalton or less, are likely to have good absorption and permeation inbiological systems and are consequently more likely to be successfuldrug candidates than compounds with a molecular weight above 500 Dalton(Lipinski et al. (1997)). Peptides comprise another preferred class ofdrug candidate compounds. Peptides may be excellent drug candidates andthere are multiple examples of commercially valuable peptides such asfertility hormones and platelet aggregation inhibitors. Naturalcompounds are another preferred class of drug candidate compound. Suchcompounds are found in and extracted from natural sources, and which maythereafter be synthesized. The lipids are another preferred class ofdrug candidate compound.

Another preferred class of drug candidate compounds is an antibody. Thepresent invention also provides antibodies directed against the TARGETS.These antibodies may be endogenously produced to bind to the TARGETSwithin the cell, or added to the tissue to bind to the TARGETpolypeptide present outside the cell. These antibodies may be monoclonalantibodies or polyclonal antibodies. The present invention includeschimeric, single chain, and humanized antibodies, as well as FAbfragments and the products of a FAb expression library, and Fv fragmentsand the products of an Fv expression library.

In certain embodiments, polyclonal antibodies may be used in thepractice of the invention. The skilled artisan knows methods ofpreparing polyclonal antibodies. Polyclonal antibodies can be raised ina mammal, for example, by one or more injections of an immunizing agentand, if desired, an adjuvant. Typically, the immunizing agent and/oradjuvant will be injected in the mammal by multiple subcutaneous orintraperitoneal injections. Antibodies may also be generated against theintact TARGET protein or polypeptide, or against a fragment, derivativesincluding conjugates, or other epitope of the TARGET protein orpolypeptide, such as the TARGET embedded in a cellular membrane, or alibrary of antibody variable regions, such as a phage display library.

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants that may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). One skilled in the art withoutundue experimentation may select the immunization protocol.

In some embodiments, the antibodies may be monoclonal antibodies.Monoclonal antibodies may be prepared using methods known in the art.The monoclonal antibodies of the present invention may be “humanized” toprevent the host from mounting an immune response to the antibodies. A“humanized antibody” is one in which the complementarity determiningregions (CDRs) and/or other portions of the light and/or heavy variabledomain framework are derived from a non-human immunoglobulin, but theremaining portions of the molecule are derived from one or more humanimmunoglobulins. Humanized antibodies also include antibodiescharacterized by a humanized heavy chain associated with a donor oracceptor unmodified light chain or a chimeric light chain, or viceversa. The humanization of antibodies may be accomplished by methodsknown in the art (see, e.g. Mark and Padlan, (1994) “Chapter 4.Humanization of Monoclonal Antibodies”, The Handbook of ExperimentalPharmacology Vol. 113, Springer-Verlag, New York). Transgenic animalsmay be used to express humanized antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter,(1991) J. Mol. Biol. 227:381-8; Marks et al. (1991) J. Mol. Biol.222:581-97). The techniques of Cole, et al. and Boerner, et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole, etal. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77; Boerner, et al (1991) J. Immunol., 147(1):86-95).

Techniques known in the art for the production of single chainantibodies can be adapted to produce single chain antibodies to theTARGETS. The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens and preferably for a cell-surface protein or receptor orreceptor subunit. In the present case, one of the binding specificitiesis for one domain of the TARGET; the other one is for another domain ofthe TARGET.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello (1983) Nature 305:537-9). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. Affinitychromatography steps usually accomplish the purification of the correctmolecule. Similar procedures are disclosed in Trauneeker, et al. (1991)EMBO J. 10:3655-9.

According to another preferred embodiment, the assay method uses a drugcandidate compound identified as having a binding affinity for theTARGET, and/or has already been identified as having down-regulatingactivity such as antagonist activity for the TARGET.

The present invention further relates to a method for increasingfunctional activity of CF-associated mutant CFTR comprising contactingsaid cells with an expression inhibitory agent comprising apolynucleotide sequence that complements at least about 15 to about 30,particularly at least 17 to about 30, most particularly at least 17 toabout 25 contiguous nucleotides of a nucleotide sequence encoding apolypeptide TARGET or portion thereof including the nucleotide sequencesselected from the group consisting of SEQ ID NO: 1-29.

Another aspect of the present invention relates to a method forincreasing the functional activity of CF-associated mutant CFTR,comprising by contacting said cell with an expression-inhibiting agentthat inhibits the translation in the cell of a polyribonucleotideencoding the TARGET. A particular embodiment relates to a compositioncomprising a polynucleotide including at least one antisense strand thatfunctions to pair the agent with the TARGET mRNA, and therebydown-regulate or block the expression of the TARGET. The inhibitoryagent preferably comprises antisense polynucleotide, a ribozyme, and asmall interfering RNA (siRNA), wherein said agent comprises a nucleicacid sequence complementary to, or engineered from, anaturally-occurring polynucleotide sequence encoding a portion of apolypeptide comprising the amino acid sequence SEQ ID NO: 30-55. In apreferred embodiment the expression-inhibiting agent is complementary toa polynucleotide sequence consisting of SEQ ID NO: 1-29. In a preferredembodiment, the nucleotide sequence is complementary to a polynucleotidecomprising a sequence selected from the group SEQ ID NO: 56-99. Inanother preferred embodiment the expression-inhibiting agent iscomplementary to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO: 56-99.

An embodiment of the present invention relates to a method wherein theexpression-inhibiting agent is selected from the group consisting ofantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 30-55, a smallinterfering RNA (siRNA, preferably shRNA,) that is sufficientlycomplementary to a portion of the polyribonucleotide coding for SEQ IDNO: 30-55, such that the siRNA, preferably shRNA, interferes with thetranslation of the TARGET polyribonucleotide to the TARGET polypeptide.Preferably the expression-inhibiting agent is an antisense RNA,ribozyme, antisense oligodeoxynucleotide, or siRNA, preferably shRNA,complementary to a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1-29. In a preferred embodiment, the nucleotidesequence is complementary to a polynucleotide comprising a sequenceselected from the group SEQ ID NO: 56-99. In another preferredembodiment, the nucleotide sequence is complementary to a polynucleotideselected from the group consisting of SEQ ID NO: 56-99.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level. Antisensenucleic acids of the invention are preferably nucleic acid fragmentscapable of specifically hybridizing with all or part of a nucleic acidencoding the TARGET or the corresponding messenger RNA. In addition,antisense nucleic acids may be designed which decrease expression of thenucleic acid sequence capable of encoding the TARGET by inhibitingsplicing of its primary transcript. Any length of antisense sequence issuitable for practice of the invention so long as it is capable ofdown-regulating or blocking expression of a nucleic acid coding for theTARGETS. Preferably, the antisense sequence is at least about 17nucleotides in length. The preparation and use of antisense nucleicacids, DNA encoding antisense RNAs and the use of oligo and geneticantisense is known in the art.

One embodiment of expression-inhibitory agent is a nucleic acid that isantisense to a nucleic acid selected from the group consisting of SEQ IDNO: 1-29. For example, an antisense nucleic acid (e.g. DNA) may beintroduced into cells in vitro, or administered to a subject in vivo, asgene therapy to inhibit cellular expression of a nucleic acid selectedfrom the group consisting of SEQ ID NO: 1-29. Antisense oligonucleotidespreferably comprise a sequence containing from about 15 to about 100nucleotides and more preferably the antisense oligonucleotides comprisefrom about 17 to about 30, most particularly at least 17 to about 25.Antisense nucleic acids may be prepared from about 10 to about 30contiguous nucleotides complementary to a nucleic acid sequence selectedfrom the sequences of SEQ ID NO: 1-29.

The skilled artisan can readily utilize any of several strategies tofacilitate and simplify the selection process for antisense nucleicacids and oligonucleotides effective in inhibition of TARGET OPGexpression. Predictions of the binding energy or calculation ofthermodynamic indices between an oligonucleotide and a complementarysequence in an mRNA molecule may be utilized (Chiang et al. (1991) J.Biol. Chem. 266:18162-18171; Stull et al. (1992) Nucl. Acids Res.20:3501-3508). Antisense oligonucleotides may be selected on the basisof secondary structure (Wickstrom et al (1991) in Prospects forAntisense Nucleic Acid Therapy of Cancer and AIDS, Wickstrom, ed.,Wiley-Liss, Inc., New York, pp. 7-24; Lima et al. (1992) Biochem.31:12055-12061). Schmidt and Thompson (U.S. Pat. No. 6,416,951) describea method for identifying a functional antisense agent comprisinghybridizing an RNA with an oligonucleotide and measuring in real timethe kinetics of hybridization by hybridizing in the presence of anintercalation dye or incorporating a label and measuring thespectroscopic properties of the dye or the label's signal in thepresence of unlabelled oligonucleotide. In addition, any of a variety ofcomputer programs may be utilized which predict suitable antisenseoligonucleotide sequences or antisense targets utilizing variouscriteria recognized by the skilled artisan, including for example theabsence of self-complementarity, the absence hairpin loops, the absenceof stable homodimer and duplex formation (stability being assessed bypredicted energy in kcal/mol). Examples of such computer programs arereadily available and known to the skilled artisan and include the OLIGO4 or OLIGO 6 program (Molecular Biology Insights, Inc., Cascade, Colo.)and the Oligo Tech program (Oligo Therapeutics Inc., Wilsonville,Oreg.). In addition, antisense oligonucleotides suitable in the presentinvention may be identified by screening an oligonucleotide library, ora library of nucleic acid molecules, under hybridization conditions andselecting for those which hybridize to the target RNA or nucleic acid(see for example U.S. Pat. No. 6,500,615). Mishra and Toulme have alsodeveloped a selection procedure based on selective amplification ofoligonucleotides that bind target (Mishra et al (1994) Life Sciences317:977-982). Oligonucleotides may also be selected by their ability tomediate cleavage of target RNA by RNAse H, by selection andcharacterization of the cleavage fragments (Ho et al (1996) Nucl AcidsRes 24:1901-1907; Ho et al (1998) Nature Biotechnology 16:59-630).Generation and targeting of oligonucleotides to GGGA motifs of RNAmolecules has also been described (U.S. Pat. No. 6,277,981).

The antisense nucleic acids are preferably oligonucleotides and mayconsist entirely of deoxyribo-nucleotides, modifieddeoxyribonucleotides, or some combination of both. The antisense nucleicacids can be synthetic oligonucleotides. The oligonucleotides may bechemically modified, if desired, to improve stability and/orselectivity. Since oligonucleotides are susceptible to degradation byintracellular nucleases, the modifications can include, for example, theuse of a sulfur group to replace the free oxygen of the phosphodiesterbond. This modification is called a phosphorothioate linkage.Phosphorothioate antisense oligonucleotides are water soluble,polyanionic, and resistant to endogenous nucleases. In addition, when aphosphorothioate antisense oligonucleotide hybridizes to its targetsite, the RNA-DNA duplex activates the endogenous enzyme ribonuclease(RNase) H, which cleaves the mRNA component of the hybrid molecule.Oligonucleotides may also contain one or more substituted sugarmoieties. Particular oligonucleotides comprise one of the following atthe 2′ position: OH, SH, SCH₃, F, OCN, heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. Similar modificationsmay also be made at other positions on the oligonucleotide, particularlythe 3′ position of the sugar on the 3′ terminal nucleotide and the 5′position of 5′ terminal nucleotide.

In addition, antisense oligonucleotides with phosphoramidite andpolyamide (peptide) linkages can be synthesized. These molecules shouldbe very resistant to nuclease degradation. Furthermore, chemical groupscan be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5)of pyrimidines to enhance stability and facilitate the binding of theantisense oligonucleotide to its target site. Modifications may include2′-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxyphosphorothioates, modified bases, as well as other modifications knownto those of skill in the art.

Another type of expression-inhibitory agent that can reduce the level ofthe TARGETS is the ribozyme. Ribozymes are catalytic RNA molecules (RNAenzymes) that have separate catalytic and substrate binding domains. Thesubstrate binding sequence combines by nucleotide complementarity and,possibly, non-hydrogen bond interactions with its target sequence. Thecatalytic portion cleaves the target RNA at a specific site. Thesubstrate domain of a ribozyme can be engineered to direct it to aspecified mRNA sequence. The ribozyme recognizes and then binds a targetmRNA through complementary base pairing. Once it is bound to the correcttarget site, the ribozyme acts enzymatically to cut the target mRNA.Cleavage of the mRNA by a ribozyme destroys its ability to directsynthesis of the corresponding polypeptide. Once the ribozyme hascleaved its target sequence, it is released and can repeatedly bind andcleave at other mRNAs.

Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitisdelta virus, group I intron or RNaseP RNA (in association with an RNAguide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing ahammerhead or hairpin structure are readily prepared since thesecatalytic RNA molecules can be expressed within cells from eukaryoticpromoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9). A ribozymeof the present invention can be expressed in eukaryotic cells from theappropriate DNA vector. If desired, the activity of the ribozyme may beaugmented by its release from the primary transcript by a secondribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).

Ribozymes may be chemically synthesized by combining anoligodeoxyribonucleotide with a ribozyme catalytic domain (20nucleotides) flanked by sequences that hybridize to the target mRNAafter transcription. The oligodeoxyribonucleotide is amplified by usingthe substrate binding sequences as primers. The amplification product iscloned into a eukaryotic expression vector.

Ribozymes are expressed from transcription units inserted into DNA, RNA,or viral vectors. Transcription of the ribozyme sequences are drivenfrom a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymeraseII (pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onnearby gene regulatory sequences. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Gao and Huang, (1993) Nucleic AcidsRes. 21:2867-72). It has been demonstrated that ribozymes expressed fromthese promoters can function in mammalian cells (Kashani-Sabet, et al.(1992) Antisense Res. Dev. 2:3-15).

A particularly preferred inhibitory agent is a small interfering RNA(siRNA, preferably shRNA). siRNA, preferably shRNA, mediate thepost-transcriptional process of gene silencing by double stranded RNA(dsRNA) that is homologous in sequence to the silenced RNA. siRNAaccording to the present invention comprises a sense strand of 15-30,particularly 17-30, most particularly 17-25 nucleotides complementary orhomologous to a contiguous 17-25 nucleotide sequence of a sequenceselected from the group consisting of SEQ ID NO: 1-29, and an antisensestrand of 17-23 nucleotides complementary to the sense strand. Exemplarysequences are described as sequences complementary to SEQ ID NO: 56-99.The most preferred siRNA comprises sense and anti-sense strands that are100 percent complementary to each other and the target polynucleotidesequence. Preferably the siRNA further comprises a loop region linkingthe sense and the antisense strand.

A self-complementing single stranded siRNA molecule polynucleotideaccording to the present invention comprises a sense portion and anantisense portion connected by a loop region linker. Preferably, theloop region sequence is 4-30 nucleotides long, more preferably 5-15nucleotides long and most preferably 12 nucleotides long. In a mostparticular embodiment the linker sequence is UUGCUAUA or GUUUGCUAUAAC(SEQ ID NO: 100). Self-complementary single stranded siRNAs form hairpinloops and are more stable than ordinary dsRNA. In addition, they aremore easily produced from vectors.

Analogous to antisense RNA, the siRNA can be modified to confirmresistance to nucleolytic degradation, or to enhance activity, or toenhance cellular distribution, or to enhance cellular uptake, suchmodifications may consist of modified internucleoside linkages, modifiednucleic acid bases, modified sugars and/or chemical linkage the siRNA toone or more moieties or conjugates. The nucleotide sequences areselected according to siRNA designing rules that give an improvedreduction of the TARGET sequences compared to nucleotide sequences thatdo not comply with these siRNA designing rules (For a discussion ofthese rules and examples of the preparation of siRNA, WO 2004/094636,and US 2003/0198627, are hereby incorporated by reference).

The present invention also relates to compositions, and methods usingsaid compositions, comprising a DNA expression vector capable ofexpressing a polynucleotide capable of increasing functional activity ofCF-associated mutant CFTR and described hereinabove as an expressioninhibition agent.

A particular aspect of these compositions and methods relates to thedown-regulation or blocking of the expression of the TARGET by theinduced expression of a polynucleotide encoding an intracellular bindingprotein that is capable of selectively interacting with the TARGET. Anintracellular binding protein includes any protein capable ofselectively interacting, or binding, with the polypeptide in the cell inwhich it is expressed and neutralizing the function of the polypeptide.Preferably, the intracellular binding protein is a neutralizing antibodyor a fragment of a neutralizing antibody having binding affinity to anepitope of a TARGET selected from the group consisting of SEQ ID NO:30-55. More preferably, the intracellular binding protein is a singlechain antibody.

A particular embodiment of this composition comprises theexpression-inhibiting agent selected from the group consisting ofantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for a TARGET selected from thegroup consisting of SEQ ID NO: 30-55, and a small interfering RNA(siRNA) that is sufficiently homologous to a portion of thepolyribonucleotide coding for a TARGET selected from the groupconsisting of SEQ ID NO: 30-55, such that the siRNA interferes with thetranslation of the TARGET polyribonucleotide to the TARGET polypeptide.

The polynucleotide expressing the expression-inhibiting agent, or apolynucleotide expressing the TARGET polypeptide in cells, isparticularly included within a vector. The polynucleic acid is operablylinked to signals enabling expression of the nucleic acid sequence andis introduced into a cell utilizing, preferably, recombinant vectorconstructs, which will express the antisense nucleic acid once thevector is introduced into the cell. A variety of viral-based systems areavailable, including adenoviral, retroviral, adeno-associated viral,lentiviral, herpes simplex viral or a sendaiviral vector systems, andall may be used to introduce and express polynucleotide sequence for theexpression-inhibiting agents or the polynucleotide expressing the TARGETpolypeptide in the target cells.

Particularly, the viral vectors used in the methods of the presentinvention are replication defective. Such replication defective vectorswill usually pack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution, partial deletion or addition ofone or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome, which are necessary for encapsidating, theviral particles.

In a preferred embodiment, the viral element is derived from anadenovirus. Preferably, the vehicle includes an adenoviral vectorpackaged into an adenoviral capsid, or a functional part, derivative,and/or analogue thereof. Adenovirus biology is also comparatively wellknown on the molecular level. Many tools for adenoviral vectors havebeen and continue to be developed, thus making an adenoviral capsid apreferred vehicle for incorporating in a library of the invention. Anadenovirus is capable of infecting a wide variety of cells. However,different adenoviral serotypes have different preferences for cells. Tocombine and widen the target cell population that an adenoviral capsidof the invention can enter in a preferred embodiment, the vehicleincludes adenoviral fiber proteins from at least two adenoviruses.Preferred adenoviral fiber protein sequences are serotype 17, 45 and 51.Techniques or construction and expression of these chimeric vectors aredisclosed in US 2003/0180258 and US 2004/0071660, hereby incorporated byreference.

In a preferred embodiment, the nucleic acid derived from an adenovirusincludes the nucleic acid encoding an adenoviral late protein or afunctional part, derivative, and/or analogue thereof. An adenoviral lateprotein, for instance an adenoviral fiber protein, may be favorably usedto target the vehicle to a certain cell or to induce enhanced deliveryof the vehicle to the cell. Preferably, the nucleic acid derived from anadenovirus encodes for essentially all adenoviral late proteins,enabling the formation of entire adenoviral capsids or functional parts,analogues, and/or derivatives thereof. Preferably, the nucleic acidderived from an adenovirus includes the nucleic acid encoding adenovirusE2A or a functional part, derivative, and/or analogue thereof.Preferably, the nucleic acid derived from an adenovirus includes thenucleic acid encoding at least one E4-region protein or a functionalpart, derivative, and/or analogue thereof, which facilitates, at leastin part, replication of an adenoviral derived nucleic acid in a cell.The adenoviral vectors used in the examples of this application areexemplary of the vectors useful in the present method of treatmentinvention.

Certain embodiments of the present invention use retroviral vectorsystems. Retroviruses are integrating viruses that infect dividingcells, and their construction is known in the art. Retroviral vectorscan be constructed from different types of retrovirus, such as, MoMuLV(“murine Moloney leukemia virus”) MSV (“murine Moloney sarcoma virus”),HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Roussarcoma virus”) and Friend virus. Lentiviral vector systems may also beused in the practice of the present invention.

In other embodiments of the present invention, adeno-associated viruses(“AAV”) are utilized. The AAV viruses are DNA viruses of relativelysmall size that integrate, in a stable and site-specific manner, intothe genome of the infected cells. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies.

In the vector construction, the polynucleotide agents of the presentinvention may be linked to one or more regulatory regions. Selection ofthe appropriate regulatory region or regions is a routine matter, withinthe level of ordinary skill in the art. Regulatory regions includepromoters, and may include enhancers, suppressors, etc.

Promoters that may be used in the expression vectors of the presentinvention include both constitutive promoters and regulated (inducible)promoters. The promoters may be prokaryotic or eukaryotic depending onthe host. Among the prokaryotic (including bacteriophage) promotersuseful for practice of this invention are lac, lacZ, T3, T7, lambdaP_(r), P₁, and trp promoters. Among the eukaryotic (including viral)promoters useful for practice of this invention are ubiquitous promoters(e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters(e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters(e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g.actin promoter in smooth muscle cells, or Flt and Flk promoters activein endothelial cells), including animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift, et al. (1984) Cell 38:639-46; Ornitz, et al. (1986)Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987)Hepatology 7:425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, (1985) Nature 315:115-22),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mousemammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95),albumin gene control region which is active in liver (Pinkert, et al.(1987) Genes and Devel. 1:268-76), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol.,5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsingene control region which is active in the liver (Kelsey, et al. (1987)Genes and Devel., 1: 161-71), beta-globin gene control region which isactive in myeloid cells (Mogram, et al. (1985) Nature 315:338-40;Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene controlregion which is active in oligodendrocyte cells in the brain (Readhead,et al. (1987) Cell 48:703-12), myosin light chain-2 gene control regionwhich is active in skeletal muscle (Sani, (1985) Nature 314.283-6), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason, et al. (1986) Science 234:1372-8).

Other promoters which may be used in the practice of the inventioninclude promoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (e.g. steroid hormone receptor,retinoic acid receptor), tetracycline-regulated transcriptionalmodulators, cytomegalovirus immediate-early, retroviral LTR,metallothionein, SV-40, E1a, and MLP promoters. Further promoters whichmay be of use in the practice of the invention include promoters whichare active and/or expressed in lung cells, or in epithelial cells,particularly in airway or brochial epithelial cells.

Additional vector systems include the non-viral systems that facilitateintroduction of polynucleotide agents into a patient. For example, a DNAvector encoding a desired sequence can be introduced in vivo bylipofection. Synthetic cationic lipids designed to limit thedifficulties encountered with liposome-mediated transfection can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Feigner, et. al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7);see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer,et al. (1993) Science 259:1745-8). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Feigner andRingold, (1989) Nature 337:387-8). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO 95/18863 and WO 96/17823, and inU.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenousgenes into the specific organs in vivo has certain practical advantagesand directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, forexample, pancreas, liver, kidney, and the brain. Lipids may bechemically coupled to other molecules for the purpose of targeting.Targeted peptides, e.g., hormones or neurotransmitters, and proteins forexample, antibodies, or non-peptide molecules could be coupled toliposomes chemically. Other molecules are also useful for facilitatingtransfection of a nucleic acid in vivo, for example, a cationicoligopeptide (e.g., International Patent Publication WO 95/21931),peptides derived from DNA binding proteins (e.g., International PatentPublication WO 96/25508), or a cationic polymer (e.g., InternationalPatent Publication WO 95/21931).

It is also possible to introduce a DNA vector in vivo as a naked DNAplasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). NakedDNA vectors for therapeutic purposes can be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem.267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al.Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30).Receptor-mediated DNA delivery approaches can also be used (Curie), etal. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem.262:4429-32).

The present invention also provides biologically compatible,CF-associated mutant CFTR inducing compositions comprising an effectiveamount of one or more compounds identified as TARGET inhibitors, and/orthe expression-inhibiting agents as described hereinabove.

A biologically compatible composition is a composition, that may besolid, liquid, gel, or other form, in which the compound,polynucleotide, vector, and antibody of the invention is maintained inan active form, e.g., in a form able to effect a biological activity.For example, a compound of the invention would have inverse agonist orantagonist activity on the TARGET; a nucleic acid would be able toreplicate, translate a message, or hybridize to a complementary mRNA ofthe TARGET; a vector would be able to transfect a target cell andexpress the antisense, antibody, ribozyme or siRNA as describedhereinabove; an antibody would bind a the TARGET polypeptide domain.

A particular biologically compatible composition is an aqueous solutionthat is buffered using, e.g., Tris, phosphate, or HEPES buffer,containing salt ions. Usually the concentration of salt ions will besimilar to physiological levels. Biologically compatible solutions mayinclude stabilizing agents and preservatives. In a more preferredembodiment, the biocompatible composition is a pharmaceuticallyacceptable composition. Such compositions can be formulated foradministration by topical, oral, parenteral, intranasal, subcutaneous,and intraocular, routes. Parenteral administration is meant to includeintravenous injection, intramuscular injection, intra-arterial injectionor infusion techniques. The composition may be administered parenterallyin dosage unit formulations containing standard, well-known non-toxicphysiologically acceptable carriers, adjuvants and vehicles as desired.

A particular embodiment of the present composition invention is apharmaceutical composition comprising a therapeutically effective amountof an expression-inhibiting agent as described hereinabove, in admixturewith a pharmaceutically acceptable carrier. Another preferred embodimentis a pharmaceutical composition for the treatment or prevention of adisease involving a decrease in functional activity of CF-associatedmutant CFTR, or a susceptibility to the condition, comprising aneffective amount of the TARGET antagonist or inverse agonist, itspharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof in admixture with a pharmaceutically acceptable carrier.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient. Pharmaceutical compositions for oral usecan be prepared by combining active compounds with solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethyl-cellulose; gums including arabic and tragacanth;and proteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification or to characterizethe quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Preferred sterile injectable preparations can be a solution orsuspension in a non-toxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (e.g. monosodium or disodium phosphate, sodium,potassium; calcium or magnesium chloride, or mixtures of such salts),Ringer's solution, dextrose, water, sterile water, glycerol, ethanol,and combinations thereof 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media. Any bland fixedoil can be employed including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

The agents or compositions of the invention may be combined foradministration with or embedded in polymeric carrier(s), biodegradableor biomimetic matrices or in a scaffold. The carrier, matrix or scaffoldmay be of any material that will allow composition to be incorporatedand expressed and will be compatible with the addition of cells or inthe presence of cells. Particularly, the carrier matrix or scaffold ispredominantly non-immunogenic and is biodegradable. Examples ofbiodegradable materials include, but are not limited to, polyglycolicacid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suturematerial, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin,alginate, cotton, or other naturally-occurring biodegradable materials.It may be preferable to sterilize the matrix or scaffold material priorto administration or implantation, e.g., by treatment with ethyleneoxide or by gamma irradiation or irradiation with an electron beam. Inaddition, a number of other materials may be used to form the scaffoldor framework structure, including but not limited to: nylon(polyamides), dacron (polyesters), polystyrene, polypropylene,polyacrylates, polyvinyl compounds (e.g., polyvinylchloride),polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox(TPX), polymers of hydroxy acids such as polylactic acid (PLA),polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA),polyorthoesters, polyanhydrides, polyphosphazenes, and a variety ofpolyhydroxyalkanoates, and combinations thereof. Matrices suitableinclude a polymeric mesh or sponge and a polymeric hydrogel. In theparticular embodiment, the matrix is biodegradable over a time period ofless than a year, more particularly less than six months, mostparticularly over two to ten weeks. The polymer composition, as well asmethod of manufacture, can be used to determine the rate of degradation.For example, mixing increasing amounts of polylactic acid withpolyglycolic acid decreases the degradation time. Meshes of polyglycolicacid that can be used can be obtained commercially, for instance, fromsurgical supply companies (e.g., Ethicon, N.J.). In general, thesepolymers are at least partially soluble in aqueous solutions, such aswater, buffered salt solutions, or aqueous alcohol solutions, that havecharged side groups, or a monovalent ionic salt thereof.

The composition medium can also be a hydrogel, which is prepared fromany biocompatible or non-cytotoxic homo- or hetero-polymer, such as ahydrophilic polyacrylic acid polymer that can act as a drug absorbingsponge. Certain of them, such as, in particular, those obtained fromethylene and/or propylene oxide are commercially available. A hydrogelcan be deposited directly onto the surface of the tissue to be treated,for example during surgical intervention.

Embodiments of pharmaceutical compositions of the present inventioncomprise a replication defective recombinant viral vector encoding thepolynucleotide inhibitory agent of the present invention and atransfection enhancer, such as poloxamer. An example of a poloxamer isPoloxamer 407, which is commercially available (BASF, Parsippany, N.J.)and is a non-toxic, biocompatible polyol. A poloxamer impregnated withrecombinant viruses may be deposited directly on the surface of thetissue to be treated, for example during a surgical intervention.Poloxamer possesses essentially the same advantages as hydrogel whilehaving a lower viscosity.

The active expression-inhibiting agents may also be entrapped inmicrocapsules prepared, for example, by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980) 16th edition, Osol, A. Ed.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

As defined above, therapeutically effective dose means that amount ofprotein, polynucleotide, peptide, or its antibodies, agonists orantagonists, which ameliorate the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED₅₀ (the dose therapeutically effective in 50% of the population)and LD₅₀ (the dose lethal to 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, and it can beexpressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions thatexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used in formulating a rangeof dosage for human use. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The pharmaceutical compositions according to this invention may beadministered to a subject by a variety of methods. They may be addeddirectly to target tissues, complexed with cationic lipids, packagedwithin liposomes, or delivered to target cells by other methods known inthe art. Localized administration to the desired tissues may be done bydirect injection, transdermal absorption, catheter, infusion pump orstent. The DNA, DNA/vehicle complexes, or the recombinant virusparticles are locally administered to the site of treatment. Alternativeroutes of delivery include, but are not limited to, intravenousinjection, intramuscular injection, subcutaneous injection, aerosolinhalation, oral (tablet or pill form), topical, systemic, ocular,intraperitoneal and/or intrathecal delivery. Examples of ribozymedelivery and administration are provided in Sullivan et al. WO 94/02595.

Antibodies according to the invention may be delivered as a bolus only,infused over time or both administered as a bolus and infused over time.Those skilled in the art may employ different formulations forpolynucleotides than for proteins. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

As discussed hereinabove, recombinant viruses may be used to introduceDNA encoding polynucleotide agents useful in the present invention.Recombinant viruses according to the invention are generally formulatedand administered in the form of doses of between about 10⁴ and about10¹⁴ pfu. In the case of AAVs and adenoviruses, doses of from about 10⁶to about 10¹¹ pfu are preferably used. The term pfu (“plaque-formingunit”) corresponds to the infective power of a suspension of virions andis determined by infecting an appropriate cell culture and measuring thenumber of plaques formed. The techniques for determining the pfu titreof a viral solution are well documented in the prior art.

In one aspect the present invention provides methods of preventingand/or treating disorders involving ER-associated protein misfolding,said methods comprising administering to a subject a therapeuticallyeffective amount of an agent as disclosed herein. In a particularembodiment, the agent is selected from an expression-inhibiting agentand an antibody.

In a further aspect the present invention provides a method ofpreventing and/or treating a disease characterized by abnormaltrafficking of a disease associated protein, said method comprisingadministering to a subject a therapeutically effective amount of anagent as disclosed herein. In a particular embodiment, the agent isselected from an expression-inhibiting agent and an antibody.

In a particular aspect, the present invention provides a method ofpreventing and/or treating Cystic Fibrosis, said method comprisingadministering to a subject a therapeutically effective amount of anagent as disclosed herein. In a particular embodiment, the agent isselected from an expression-inhibiting agent and an antibody.

A further aspect of the invention relates to a method of treating orpreventing a disease involving a decrease in CF-associated mutant CFTRfunction, comprising administering to said subject a therapeuticallyeffective amount of an agent as disclosed herein. In a particularembodiment, the agent is selected from an expression-inhibiting agentand an antibody.

The invention also relates to the use of an agent as described above forthe preparation of a medicament for treating or preventing a diseaseinvolving ER-associated protein misfolding. In a particular embodiment,the disease is characterised by abnormal trafficking of adisease-associated protein. In a particular embodiment of the presentinvention the disease is selected from Cystic Fibrosis, Parkinson'sDisease, Gaucher's Disease, Nephrogenic diabetes insipidus, Emphysemaand Liver Disease (alpha-1-antitrypsin deficiency), Maple syrup urinedisease, Fabry's disease, Hypogonadotropic hypogonadism,Hyperinsulinemic hypoglycemia, beta-Galactosidosis, Wilson disease, LongQT syndrome, Retinitis pigmentosa, transthyretin-linked amyloidosis,Alzheimer's Disease, Prion disease, and inclusion body myositis. In afurther embodiment of the present invention the disease is cysticfibrosis.

The invention also relates to the use of an agent as described above forthe preparation of a medicament for treating or preventing an airwayepithelial or brochial inflammatory disease, including asthma or COPD.

The present invention also provides a method of treating and/orpreventing a disease involving ER-associated protein misfolding saidmethod comprising administering, to a subject suffering from, orsusceptible to, a disease involving ER-associated protein misfolding, apharmaceutical composition or compound as described herein, particularlya therapeutically effective amount of an agent which inhibits theexpression or activity of a TARGET as identified herein. In oneembodiment, the disease is characterized by abnormal trafficking of adisease-associated protein. In a further embodiment the disease isselected from cystic fibrosis, Parkinson's disease, Gaucher's disease,nephrogenic diabetes insipidus, emphysema and liver disease(alpha-1-antitrypsin deficiency), Maple syrup urine disease, Fabry'sdisease, hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia,beta-galactosidosis, Wilson's disease, long QT syndrome, retinitispigmentosa, transthyretin-linked amyloidosis, Alzheimer's Disease, Priondisease, and inclusion body myositis. In a further embodiment of thepresent invention the disease is cystic fibrosis.

The present invention also provides a method of treating and/orpreventing asthma and COPD said method comprising administering, to asubject suffering from or susceptible to, asthma and COPD apharmaceutical composition or an agent as described herein.

The invention also relates to an agent or a pharmaceutical compositionas described above for use in the treatment and/or prevention of adisease involving ER-associated protein misfolding. In a particularembodiment, the disease is characterised by abnormal trafficking of adisease-associated protein. In a particular embodiment of the presentinvention the disease is selected from cystic fibrosis, Parkinson'sdisease, Gaucher's disease, nephrogenic diabetes insipidus, emphysemaand liver disease (alpha-1-antitrypsin deficiency), Maple syrup urinedisease, Fabry's disease, hypogonadotropic hypogonadism,hyperinsulinemic hypoglycemia, beta-galactosidosis, Wilson's disease,long QT syndrome, retinitis pigmentosa, transthyretin-linkedamyloidosis, Alzheimer's disease, Prion disease, and inclusion bodymyositis. In a further embodiment of the present invention the diseaseis cystic fibrosis.

The invention also relates to an agent or a pharmaceutical compositionas described above for use in the treatment and/or prevention of anairway epithelial or brochial inflammatory disease, including asthma orCOPD.

Administration of the agent or pharmaceutical composition of the presentinvention to the subject patient includes both self-administration andadministration by another person. The patient may be in need oftreatment for an existing disease or medical condition, or may desireprophylactic treatment to prevent or reduce the risk for diseases andmedical conditions characterized by ER-associated protein misfolding.The agent of the present invention may be delivered to the subjectpatient orally, transdermally, via inhalation, injection, nasally,rectally or via a sustained release formulation.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving ER-associated protein misfolding,comprising determining the amount of a polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 30-55 ina biological sample, and comparing the amount with the amount of thepolypeptide in a healthy subject, wherein an increase of the amount ofpolypeptide compared to the healthy subject is indicative of thepresence of the pathological condition. In one embodiment, the diseaseis characterized by abnormal trafficking of a disease-associatedprotein. In a further embodiment the disease is selected from cysticfibrosis, Parkinson's disease, Gaucher's disease, nephrogenic diabetesinsipidus, emphysema and liver disease (alpha-1-antitrypsin deficiency),Maple syrup urine disease, Fabry's disease, hypogonadotropichypogonadism, hyperinsulinemic hypoglycemia, beta-galactosidosis,Wilson's disease, long QT syndrome, retinitis pigmentosa,transthyretin-linked amyloidosis, Alzheimer's disease, Prion disease,and inclusion body myositis. In a particular embodiment, said method maybe used to diagnose a decrease in CF-associated mutant CFTRfunctionality or a susceptibility to the condition in a subject. In afurther embodiment of the present invention the disease is cysticfibrosis.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving ER-associated protein misfolding,comprising determining the activity of a polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 30-55 ina biological sample, and comparing the activity with the activity of thepolypeptide in a healthy subject, wherein an increase of the activity ofpolypeptide compared to the healthy subject is indicative of thepresence of the pathological condition. In one embodiment, the diseaseis characterized by abnormal trafficking of a disease-associatedprotein. In an embodiment, the disease is characterized by abnormalfolding of a disease-associated protein. In an embodiment, the diseaseis characterized by misfolding and degradation of a disease-associatedprotein. In a further embodiment the disease is selected from cysticfibrosis, Parkinson's disease, Gaucher's disease, nephrogenic diabetesinsipidus, emphysema and liver disease (alpha-1-antitrypsin deficiency),Maple syrup urine disease, Fabry's disease, hypogonadotropichypogonadism, hyperinsulinemic hypoglycemia, beta-galactosidosis,Wilson's disease, long QT syndrome, retinitis pigmentosa,transthyretin-linked amyloidosis, Alzheimer's disease, Prion disease,and inclusion body myositis. In a particular embodiment, said method maybe used to diagnose a decrease in CF-associated mutant CFTRfunctionality or a susceptibility to the condition in a subject. In afurther embodiment of the present invention the disease is cysticfibrosis.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving ER-associated protein misfolding,comprising determining the nucleic acid sequence of at least one of thegenes of SEQ ID NO: 1-29 within the genomic DNA of a subject; comparingthe sequence with the nucleic acid sequence obtained from a databaseand/or a healthy subject; and identifying any difference(s) related tothe onset or prevalence of the pathological conditions disclosed herein.

The polypeptides or the polynucleotides of the present inventionemployed in the methods described herein may be free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellularly. To perform the methods it is feasible to immobilizeeither the polypeptide of the present invention or the compound tofacilitate separation of complexes from uncomplexed forms of thepolypeptide, as well as to accommodate automation of the assay.Interaction (e.g., binding of) of the polypeptide of the presentinvention with a compound can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and microcentrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows thepolypeptide to be bound to a matrix. For example, the polypeptide of thepresent invention can be “His” tagged, and subsequently adsorbed ontoNi-NTA microtitre plates, or ProtA fusions with the polypeptides of thepresent invention can be adsorbed to IgG, which are then combined withthe cell lysates (e.g., ⁽³⁵⁾S-labelled) and the candidate compound, andthe mixture incubated under conditions favorable for complex formation(e.g., at physiological conditions for salt and pH). Followingincubation, the plates are washed to remove any unbound label, and thematrix is immobilized. The amount of radioactivity can be determineddirectly, or in the supernatant after dissociation of the complexes.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of the protein binding to theprotein of the present invention quantitated from the gel using standardelectrophoretic techniques.

Other techniques for immobilizing protein on matrices can also be usedin the method of identifying compounds. For example, either thepolypeptide of the present invention or the compound can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated proteinmolecules of the present invention can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the polypeptides of the presentinvention but which do not interfere with binding of the polypeptide tothe compound can be derivatized to the wells of the plate, and thepolypeptide of the present invention can be trapped in the wells byantibody conjugation. As described above, preparations of a labeledcandidate compound are incubated in the wells of the plate presentingthe polypeptide of the present invention, and the amount of complextrapped in the well can be quantitated.

The polynucleotides encoding the TARGET polypeptides are identified asSEQ ID NO: 1-29. The present inventors show herein that transfection ofmammalian cells with Ad-siRNAs targeting these genes increases thefunctional activity of CF-associated mutant CFTR.

The invention is further illustrated in the following figures andexamples.

EXPERIMENTAL SECTION Example 1 Development of a High-ThroughputScreening Method for CFTR-Dependent Halide Flux 1.1 Principal of theAssay

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), by itschloride channel function, plays a key role in chloride secretion andwater balance in epithelia throughout the body. Other halides such asiodide also make use of CFTR. Accordingly, an assay to monitorCFTR-halide flux by using a reporter protein, halide-sensitivefluorescent protein YFP, is developed to measure the functional activityof CFTR. Cells expressing this reporter protein show enhancedfluorescence quenching of YFP by extracellular isomolar iodide solutionsin the presence of activated CFTR. This is caused by the increased fluxof iodide across the plasma membrane by CFTR. The fluorescence quenchingis measured on a fluorescence plate reader.

1.2 Development of the Assay

Human lung epithelial cells are isolated from a ΔF508-CFTR patient,obtained after informed consent. These cells are stably transfected withΔF508-CFTR expression plasmids and are named CFBE41o-cells (Gruenert etal., 2004). This is a preferred cell model because it is of human originand derived from the primary organ suffering from the effect of theCF-associated mutations. Targets identified in human model systems arecommonly considered to have lower attrition as compared to targetsidentified in models from different species, which have naturallydiverged from humans during evolution. CFBE41o-cells are cultured ontissue culture grade plastic, coated with 0.1 mg/mL bovine serum albumin(BSA), 0.03 mg/mL bovine collagen type 1 and 0.01 mg/mL humanfibronectin. CFBE41o-cells are cultured in MEM containing 10% FetalBovine Serum, 2 mM glutamine, 100 IU/mL penicilline, 0.1 mg/mlstreptomycine sulfate and 0.5 mg/mL hygromycin B at 37° C., 5% CO₂ in ahumidified chamber. For high-throughput screening, 96-well plates areseeded with 1,000 cells per well.

As discussed above, measuring halide channel activity in cellsexpressing CFTR represents the preferred method for measuring thefunctional activity of CF-associated mutant CFTR. Halide channelactivity is measured using the reporter, halide-sensitive fluorescentprotein YFP (Galietta et al., 2001a).

To efficiently express the halide-sensitive fluorescent protein YFP inCFBE41o-cells, the reporter cDNA is synthesized and cloned in adenoviraladapter plasmids. dE1/dE2A (deleted for adenoviral genes E1 and E2A)adenoviruses are generated from these adapter plasmids byco-transfection of the helper plasmid pWEAd5AflII-rITR.dE2A in PER.E2Apackaging cells, as described in WO99/64582.

In order to specifically assess the activity of ΔF508-CFTR, this proteinis expressed, or as a positive control, the wild-type CFTR is expressed,from adenoviral vectors. ΔF508-CFTR cDNA or the wild-type CFTR cDNA(GenBank accession number NM_(—)000492) (SEQ ID NO: 101) is cloned inadenoviral adapter plasmids to produce adenoviral vectors.

To determine the optimal conditions for adenoviral transduction, severalconditions for the expression of the YFP halide reporter are tested. Anexperiment is performed where increasing amounts of adenoviral vectorsas defined by virus particles per cell (VPU) are used to transduceCFBE41o-cells. VPU is determined by quantitative PCR, and is defined asadenoviral particles per ml according to (Ma et al., 2001). Three daysafter transduction of the fluorescent halide reporter, transductionefficiency is measured using fluorescent activated cell sorting (FACS)(Becton Dickinson FACScalibur) with excitation at 488 nm. The outcome ofsuch an experiment is shown in FIGS. 1A and 1B. In this experiment,CFBE41o-cells are transduced with increasing VPU of adenovirus without acDNA (empty), enhanced Green Fluorescent Protein (eGFP) as a positivecontrol, and the YFP halide-sensitive fluorescent protein (YFP). Threedays after transduction, cells are detached with trypsin, fixed andanalysed with FACS (10,000 cells are counted).

As can be seen in FIGS. 1A and 1B, adenovirus without a cDNA representsthe background fluorescence in the cells (2.14% at VPU 500 and 2.85% atVPU 2000). eGFP transduction results in 93.3% and 97.4% positive cellsat VPU 500 and VPU 2000 respectively. YFP transduction results in 84.2%and 93.4% positive cells at VPU 500 and VPU 2000 respectively. YFPtransduction at VPU 2000 results in significantly stronger fluorescentsignal compared to VPU 500 (62.6 versus 27.5 respectively). Thus,transduction with the adenoviral YFP reporter at VPU 2000 is thepreferred method.

Example 2 Validation of the CFTR-Dependent Halide Flux Assay

In this example, it is shown that the halide-sensitive reporterexpressed in CFBE41o-cells can monitor functional activity of CFTR.CFBE41o-cells are transduced with the YFP fluorescent halide reporteradenoviral vector at a VPU of 2000 viral particles per cell, togetherwith adenoviral vectors expressing ΔF508-CFTR, or as a positive controlthe wild-type CFTR. Three days after transduction, wells are washed twotimes with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10mM Na₂HPO₄, 1.76 mM KH₂PO₄ at pH 7.4), and incubated in 40 microliter ofPBS containing 10 microM forskolin and 100 microM genistein for 5minutes Forskolin and genistein have been shown to activate CFTRactivity (Hwang et al., 1997), and are used here to pre-activate anyexisting CFTR. Plates are read in a fluorescent plate reader, equippedwith injectors for the delivery of reagents to the well (Perkin-ElmerEnvision 2102). Each well is read for 2 seconds at 485/530 nm(excitation/emission) prior to the addition of 110 microliteriodide-containing buffer (137 mM NaI, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mMKH₂PO₄ at pH 7.4). Fluorescent reading is continued for an additional 12sec, sampling every 200 msec.

FIG. 2 shows results of such an experiment. Injection of PBS does notquench YFP fluorescence in ΔF508-CFTR expressing cells. In contrast,injection of iodide results in a slow quenching of fluorescence, wherethe slope of the curve indicates the rate of halide flux. A minimalincrease in halide flux is observed after pre-incubation with forskolinand genistein, indicating that ΔF508-CFTR is activated by thiscombination. Furthermore, a much stronger increase in YFP fluorescencequenching is observed when the wild-type CFTR protein is expressed.These results confirm that this assay measures CFTR-dependent halideflux, and that the ΔF508-CFTR has reduced activity compared to thewild-type protein. Thus, quenching of YFP in the presence of ΔF508-CFTRis a specific measure of CFTR activity and can be used to identifycorrectors of defect ΔF508 CFTR activity in a high throughput screen.

Example 3 Screening of 11330 “Ad-siRNAs” in the CFTR-Dependent HalideFlux Assay

The CFTR-Dependent Halide Flux Assay, the development of which isdescribed in Example 1, has been screened against an arrayed collectionof 11,330 different recombinant adenoviruses mediating the expression ofshRNAs in CFBE41o-cells. These shRNAs cause a reduction in expressionlevels of genes that contain homologous sequences by a mechanism knownas RNA interference (RNAi). The 11330 Ad-siRNAs contained in the arrayedcollection target 5046 different transcripts. On average, everytranscript is targeted by 2 to 3 independent Ad-siRNAs. The screeningassay followed the following time-course: CFBE41o-cells were seeded at1000 cells per well in 96-well plates (transparent bottom, black sides).One day after seeding, an aliquot of the Ad-siRNA was applied to eachwell. Four days after seeding, each well received an aliquot ofadenovirus expressing ΔF508-CFTR and an aliquot of adenovirus expressingYFP. Seven days after seeding, the YFP assay was performed as describedin Example 2.

For every batch of Ad-siRNA plates, control plates were screened thatcontain control viruses that are produced under the same conditions asthe SilenceSelect® adenoviral collection. The viruses include sets ofnegative control viruses (N1 (Ad5-empty_KD)), N2 (Ad5-empty_KD), N3 (novirus)), together with positive control viruses (P1 (Ad5-STX8_v5_KD)),P2 (Ad5-STX8_v5_KD), P3 (Ad5-BCAP31_v3_KD), P4 (Ad5-BCAP31_v3_KD)), P5(Ad5-CFTR_v5_KI)). Every well of a virus plate contains 150 μL of viruscrude lysate. A representative example of the performance of a platetested with the screening protocol described above is shown in FIG. 3.In this figure, the calculated relative 12 sec endpoint ofiodide-mediated quenching of the YFP reporter detected upon performingthe assay for every recombinant adenovirus on the plate is shown (asdefined by the average of the last three data-points divided by theaverage of the pre-injection baseline fluorescence).

For analysis of the screening of 11,330 Ad-siRNAs, data from thefluorescent plate reader is exported and analyzed using perl scripts andthe R statistical package as follows:

-   -   1) Mean baseline fluorescence (prior to iodide injection) is        calculated (YFP expression level).    -   2) Data are normalized against the baseline (set at 1).    -   3) Wells are excluded when more than 10% of the data points are        above baseline.    -   4) Based on the input parameters, the curve fit is performed        using the R statistics program for analysis. It will include the        baseline at t=tinj, and use the rest of the data points until        t=14 s.    -   5) The exponential decay (y=a*e−bx+c) curve fit requires initial        values for variables. Therefore, the script loops through        different combinations of initial values, until it finds the        optimal curve fit.    -   6) The slope at t=tinj is returned by determining the derivative        of the function (initial slope).    -   7) The average of the three last data points (t=14 sec) is        determined and calculated relative to baseline (end-point). This        value ranges from 1 (no quenching) to 0 (complete quenching of        YFP).

Identification of hits was performed both on the calculated 12-secendpoint and the calculated initial slope. These values were expressedin fold standard deviation of the samples on the 96-well plate relativeto the mean of the samples on the 96-well plate. When either of thesevalues exceeds the cutoff value (defined as 1.5 fold the standarddeviation below the sample mean), a Ad-siRNA virus is marked as a hit.An overview of the screening data is shown in FIG. 4, with hits below−1.5. The screen of 11,330 Ad-siRNAs procedure yielded 753 hits.

Example 4 Rescreen of the Primary Hits Using Independent RepropagationMaterial

To confirm the results of the identified Ad-siRNA in the CFTR-DependentHalide Flux Assay, the following approach may be taken: the Ad-siRNAhits are repropagated using PerC6 cells (Crucell, Leiden, TheNetherlands) at a 96-well plate level, followed by retesting in theCFTR-Dependent Halide Flux Assay. First, tubes containing the crudelysates of the identified hit Ad-siRNA's samples are picked from theSilenceSelect® collection and rearranged in 96 well plates together withnegative/positive controls. As the tubes are labeled with a barcode(Screenmates™, Matrix technologies), quality checks are performed on therearranged plates. To propagate the rearranged hit viruses, 40,000PerC6.E2A cells are seeded in 200 microL of DMEM containing 10% non-heatinactivated FBS into each well of a 96 well plate and incubatedovernight at 39° C. in a humidified incubator at 10% CO₂. Subsequently,2 microL of crude lysate from the hit Ad-siRNA's rearranged in the 96well plates as indicated above is added to the PerC6.E2A cells using a96 well dispenser. The plates may then be incubated at 34° C. in ahumidified incubator at 10% CO₂ for 5 to 10 days. After this period, therepropagation plates are frozen at −20° C., provided that complete CPE(cytopathic effect) could be seen. The propagated Ad-siRNAs arerescreened in the CFTR-Dependent Halide Flux Assay.

Data analysis for each of the rescreen is performed as follows. Forevery plate the average and standard deviation is calculated for thenegative controls and may be used to convert each data point into a“cutoff value” that indicates the difference between the sample and theaverage of all negatives in terms of standard deviation of allnegatives. Threshold settings for the rescreen were −2 fold standarddeviation from the mean of the negative controls. At this cut-off, 315Ad-siRNAs are again positive in the CFTR-Dependent Halide Flux Assay.Data for the TARGETs of the present invention are shown in Table 3below, the halide flux is expressed as the fold stdev from the mean ofthe negative controls.

TABLE 3 Efficacy of the restoration of CFTR-dependent halide flux inCFBE41o- relevant to the present expression-inhibitory agent inventionForskolin-Genistein TARGET induced halide flux Gene Symbol SEQ ID NO:DNA in CFBE41o- UGT3A2 2 −5.485 PHGDH 3 −6.495 B3GNT3 4 −4.495 PPIH 5−11.49 CELSR3 6 −2.16 MC2R 7 −9.81 MAS1L 8 −3.93 LRRK2 9 −5.865 NLRP110, 11, 12, 13, 14 −7.195 PMS1 15, 16, 17 −5.065 MAK 18 −4.645 CPD 19−7.04 CST7 20 −3.87 DUSP5 21 −3.39 PTPRG 22 −4.41 IL6R 23, 24 −5.595 GHR25 −4.425 CSF3 26, 27, 28 −6.36 SPNS1 29 −7.025 STX8_v5 positive control−6.9963 BCAP31_v3 positive control −5.45241 wild-type CFTR_v5 positivecontrol −25.1772

A quality control of target Ad-siRNAs is performed as follows: TargetAd-siRNAs are propagated using derivatives of PERC6.E2A cells (Crucell,Leiden, The Netherlands) in 96-well plates, followed by sequencing thesiRNAs encoded by the target Ad-siRNA viruses. PERC6.E2A cells areseeded in 96 well plates at a density of 40,000 cells/well in 180 μLPER.E2A medium. Cells are then incubated overnight at 39° C. in a 10%CO₂ humidified incubator. One day later, cells are infected with 1 μL ofcrude cell lysate from SilenceSelect® stocks containing targetAd-siRNAs. Cells are incubated further at 34° C., 10% CO₂ untilappearance of cytopathic effect (as revealed by the swelling androunding up of the cells, typically 7 days post infection). Thesupernatant is collected, and the virus crude lysate is treated withproteinase K by adding to 4 μL Lysis buffer (1× Expand High Fidelitybuffer with MgCl2 (Roche Molecular Biochemicals, Cat. No 1332465)supplemented with 1 mg/mL proteinase K (Roche Molecular Biochemicals,Cat No 745 723) and 0.45% Tween-20 (Roche Molecular Biochemicals, Cat No1335465) to 12 μL crude lysate in sterile PCR tubes. These tubes areincubated at 55° C. for 2 hours followed by a 15 minutes inactivationstep at 95° C. For the PCR reaction, 1 μL lysate is added to a PCRmaster mix composed of 5 μL 10× Expand High Fidelity buffer with MgCl₂,0.5 μL at of dNTP mix (10 mM for each dNTP), 1 μL of “Forward primer”(10 mM stock, sequence: 5′ CCG TTT ACG TGG AGA CTC GCC 3′ (SEQ. ID NO:102), 1 μL of “Reverse Primer” (10 mM stock, sequence: 5′ CCC CCA CCTTAT ATA TAT TCT TTC C 3′) (SEQ. ID NO: 103), 0.2 μL of Expand HighFidelity DNA polymerase (3.5 U/μL, Roche Molecular Biochemicals) and41.3 μL of H2O. PCR is performed in a PE Biosystems GeneAmp PCR system9700 as follows: the PCR mixture (50 μL in total) is incubated at 95° C.for 5 minutes; each cycle runs at 95° C. for 15 sec., 55° C. for 30sec., 68° C. for 4 minutes, and is repeated for 35 cycles. A finalincubation at 68° C. is performed for 7 minutes. For sequencinganalysis, the siRNA constructs expressed by the target adenoviruses areamplified by PCR using primers complementary to vector sequencesflanking the SapI site of the pIPspAdapt6-U6 plasmid. The sequence ofthe PCR fragments is determined and compared with the expected sequence.All sequences are found to be identical to the expected sequence.

Example 5 Analysis of the Expression Levels for Certain TargetsIdentified in Human Primary Bronchial Epithelial Cells and Human Lung

Expression levels for certain identified targets are determined indifferent isolates of lung epithelial cells as follows.

Microarray data from human lung large airway epithelia, non-smoker,non-COPD (Carolan et al., 2006) is downloaded from the NCBI website(http://www.ncbi.nlm.nih gov/entrez/query.fcgi?db=gds&term=GSE5060[Accession]&cmd=search) and analyzed for expression of the HITS. Hitsexpressed in each of the samples present on these arrays (p<0.05) areconsidered expressed. All other hits are subsequently analyzed usingreal-time gene expression analysis as follows.

Two RNA samples from human total lung (either adult or fetal) areobtained from a commercial supplier (Stratagene). These samples will bereferred to as “human lung”.

Cultured primary bronchial epithelial cell isolates are obtained fromCell Applications Inc. (#502-05a, cryopreserved at first passage), fromthe University of Genova (Galieta lab, Genove, Italy) or from culturedCFBE41o-cells (human lung epithelial cells stably transfected withΔF508-CFTR expression plasmids as described above) are utilized. TotalRNA is extracted using the “RNAeasy Total RNA Isolation kit” (Qiagen).

The concentration of RNA in each sample is fluorimetrically quantified.A similar amount of RNA from each preparation is reverse transcribedinto first strand cDNA with the “Taqman reverse transcription kit” fromApplied Biosystems. Briefly, 300 ng RNA is included per 50 μL reactionmix containing 125 pmol of random hexamers, 25 U Rnase inhibitor, 62.5 UMultiscribe reverse transcriptase, 5 mM MgCl₂ and 0.5 mM of each dNTP.The reaction mixture is incubated at 25° C. for 10 minutes, followed by30 minutes incubation at 48° C. and heat inactivation (5 minutes 95° C.)of the reverse transcriptase in a thermocycler (Dyad, MJ Research).Reactions are immediately chilled to 4° C. at the end of the program. Toavoid multiple freeze/thaw cycles of the obtained cDNA, the differentsamples are pooled in 96-well plates, aliquoted and stored at −20° C.

Real-time PCR reactions are performed and monitored using the “ABI PRISM7000 Sequence Detection System Instrument” (Applied Biosystems).Pre-designed, gene-specific Taqman probe and primer sets forquantitative gene expression are purchased from Applied Biosystems aspart of the “Assays on Demand” Gene expression products. Thesecommercially available kits are quality checked by the supplier andallow quantitative determination of the amount of target cDNA in thesample. The “Assays on Demand” gene expression products are usedaccording to the protocol delivered by the supplier. The PCR mixtureconsisted of 1×“Taqman Universal PCR Mastermix no AmpErase UNG” and 1×“Taqman Gene Expression Assay on Demand mix” and 5 uL of theretro-transcription reaction product (1-40 ng of RNA converted intocDNA) in a total volume of 25 uL. After an initial denaturation step at95° C. for 10 minutes, the cDNA products are amplified with 40 cyclesconsisting of 95° C. for 15 sec, and 60° C. for 1 minute. To normalizefor variability in the initial quantities of cDNA between differentsamples, amplification reactions with the same cDNA are performed forthe housekeeping gene GAPDH using the pre-developed “Assays on demand”primer set and Taqman probe mix and “Taqman Universal PCR Mastermix”(all Applied Biosystems) according to the manufacturer's instructions.Threshold cycle values (Ct), for example, the cycle number at which theamount of amplified gene of interest reached a fixed threshold aredetermined for each sample. A HIT is considered as expressed if the Ctvalue obtained for this hit is lower than 35 in at least one of theavailable human lung isolate and at least one of the cultured humanbronchial epithelial (HBE) samples. This analysis of 315 hits yielded210 genes expressed in bronchial epithelium.

TABLE 4 Expression of the targets in lung epithelial tissue TARGET lungHBE CFBE Gene SEQ ID microarray Q-PCR Q-PCR Q-PCR Symbol NO: DNA(p-value) (Ct) (Ct) (Ct) UGT3A2 2 N/A 37.255 38.18667 33.89 PHGDH 30.030273 N/A N/A N/A B3GNT3 4 0.00415 N/A N/A N/A PPIH 5 0.00415 N/A N/AN/A CELSR3 6 0.081337 32.895 32.03 29.14 MC2R 7 0.466064 35.565 40 31.695 MAS1L 8 0.760937  33.2125 40  30.605 LRRK2 9 0.303711 27.43533.435 35.35 NLRP1 10, 11, 12, 0.888428 28.195 27.60667 27.97 13, 14PMS1 15, 16, 17 0.000244 N/A N/A N/A MAK 18 0.000244 N/A N/A N/A CPD 190.000244 N/A N/A N/A CST7 20 0.533936 26.355 40 31.32 DUSP5 21 0.000244N/A N/A N/A PTPRG 22 0.030273 N/A N/A N/A IL6R 23, 24 0.00415 N/A N/AN/A GHR 25 0.001953 N/A N/A N/A CSF3 26, 27, 28 0.544587 31.705 30.0137.66 SPNS1 29 0.018555 N/A N/A N/A

Example 6 “On Target Analysis” Using KD Viruses

To strengthen the validation of a hit, it is helpful to recapitulate itseffect using a completely independent siRNA targeting the same targetgene through a different sequence. This analysis is called the “ontarget analysis”. In practice, this was done by designing multiple newshRNA oligonucleotides against the target using a specialised algorithmpreviously described, and incorporating these into adenoviruses,according to WO 03/020931. After virus production, these viruses werearrayed in 96 well plates, together with positive and negative controlviruses. On average, 6 new independent Ad-siRNA's have been produced fora set of targets. One independent repropagation of these virus plateswas then performed as described above for the rescreen in Example 4. Theplates produced in this repropagation was tested in biological duplicatein the YFP assay at 3 MOIS according to the protocol described (Example2). Ad-siRNA's mediating an increase in the quenching of the YFPreporter above the set cutoff value in at least 1 were nominated as hitsscoring in the “on target analysis”. The cutoff value in theseexperiments was defined as the average over the negative controls+2times the standard deviation over the negative controls. Through thisexercise, 141 hits were identified with at least two active shRNAs(range: 2-6, average: 2.46). These hits are considered “on target”, andproceeded to the next validation experiment.

Example 7 Analysis of the Cell-Surface Expression of ΔF508 CFTR

A further validation of the correction of ΔF508 CFTR is the expressionof this protein on the cell surface as measured by cell-surfacebiotinylation (Prince et al., 1994). This analysis allows a morequantitative measurement of the levels of restoration of cell-surfaceexpressed ΔF508 CFTR, as well as the glycosylation status of the ΔF508CFTR protein (Cheng et al., 1990). A preferred effect of a HIT would beincreased cell-surface expression of ΔF508 CFTR, and especially “band C”(Cheng et al., 1990), fully glycosylated ΔF508 CFTR. The assay tomeasure cell surface expression of ΔF508-CFTR is performed in thefollowing fashion: CFBE41o-cells are seeded in 60 mm cell culture dishescoated with 0.1 mg/mL bovine serum albumin (BSA), 0.03 mg/mL bovinecollagen type 1 and 0.01 mg/mL human fibronectin. CFBE41o-cells arecultured in MEM containing 10% Fetal Bovine Serum, 2 mM glutamine, 100IU/mL penicillin, 0.1 mg/mL streptomycine sulfate and 0.5 mg/mLhygromycin B at 37° C., 5% CO₂ in a humidified chamber. One day afterseeding, an aliquot of the Ad-siRNA is applied to each well. Four daysafter seeding, each well receives an aliquot of adenovirus expressingΔF508-CFTR. Seven days after seeding, the cell cultures are exposed to10 microM forskolin and 100 microM genistein for 15 min at 37° C. Thecells are washed three times in PBS pH 8 to which 1 mM MgCl₂ and 0.1 mMCaCl₂ are added at 0° C., and 1.5 ml of 0.5 mg/mL sulfo-NHS-SS-biotin(Pierce #21328) diluted in PBS pH 8 supplemented with 1 mM MgCl₂ and 0.1mM CaCl₂ is added to the cell cultures. The cell culture dishes aregently rocked for 30 min at 4° C. Cell cultures are washed three timesin PBS containing 1% bovine serum albumin at 0° C. and one with PBS at0° C. Cells are scraped from the plastic in PBS at 0° C. and transferredto 1.5 mL Eppendorf tubes. The cells are harvested by centrifugation at4° C., 20,000 g for 1 min. Cells are lysed in 270 microl of RIPA buffer(1% Triton X100, 150 mM NaCl, 25 mM Tris-Cl pH7.4, 0.005% sodiumdeoxycholate, protease inhibitor cocktail (Roche #11873580001) at 2mg/mL and 0.3 mM Pefablock SC (Roche #11429868001)) for 15 min at 0° C.After centrifugation for 20 min at 20,000 g, the supernatant istransferred to an Eppendorf tube containing 30 microL of pre-washed 50%v/v NeutrAvidin agarose resin beads (Thermo Scientific #29200) in RIPAbuffer. The supernatant is incubated with the avidin beads for 16 hrs at4° C. with gentle rocking. Beads are harvested by centrifugation at 4°C., 20,000 g for 20 sec and washed twice with RIPA buffer, twice with(25 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% Triton X100) at 0° C. and twicewith (25 mM Tris-Cl pH 7.4, 150 mM NaCl) at 0° C. Beads are harvested bycentrifugation at 4° C., 20,000 g for 20 sec and resuspended in 15microl of (24 mM Tris-Cl pH 6.8, 4% glycerol, 50 mM dithiotreitol, 0.04%bromophenol-blue) and incubated for 20 min at 37° C. The supernatant isanalyzed on Western blots (BioRad Criterion XT gels, 3-8% polyacrylamide#3450131). Western blots are probed with a rat antibody raised againstCFTR (monoclonal 3G11), an antibody against a protein not expressed onthe cell surface as a negative control (anti laminA, Sigma #L1293) andan antibody against a protein constitutively expressed on the cellsurface as a positive control (E-cadherin, Abcam #ab1416). Secondaryantibodies are: ECLTM anti-rabbit IgG, HRP-Linked whole Ab (from donkey)(GE Healthcare #NA934-1), ImmunoPure Goat Anti-Rat IgG, HRP conjugated(Thermo Scientific #31470), ImmunoPure Goat Anti-Mouse IgG, HRPconjugated (Thermo Scientific #31430). Development of the blots isperformed with enhanced chemiluminescence on a Biorad ChemiDoc XRS.Quantification of the cell surface expression of ΔF508 CFTR wasperformed with Biorad Quantity One software.

Positive and negative controls for the biotinylation include incubationof cell culture at 27° C. for 48 hrs to correct misfolding andtrafficking of ΔF508 CFTR, and omission of Ad-siRNAs respectively. Nosignal is detected when the biotinylation reagent was omitted. The cellsurface expression of ΔF508 CFTR is quantitated relative to the signalobtained without Ad-siRNAs (relative to the E-cadherin signal, and setat 0) and the signal obtained at 27° C. for 48 hrs (relative to theE-cadherin signal, and set at 1). Quantification is performed both forband B ΔF508 CFTR (core glycosylated) and band C ΔF508 CFTR (fullyglycosylated). The analysis of 142 hits yields 19 TARGETS that showexpression of band C ΔF508 CFTR and band B ΔF508 CFTR on the cellsurface of CFBE41o-cells upon Ad-siRNAs-mediated knock-down of thatTARGET. These TARGETS are listed in Table 1.

TABLE 5 Cell surface exprerssion of CFTR ΔF508 in CFBE41o- cell cultureupon TARGET Ad-siRNA application CFTRΔF508 C-band/ TARGET Gene SEQ IDNO: cell surface B-band Symbol DNA expression ratio UGT3A2 2 0.906 0.278PHGDH 3 0.573 0.173 B3GNT3 4 0.113 0.121 PPIH 5 0.408 0.176 CELSR3 62.179 0.213 MC2R 7 1.082 0.412 MAS1L 8 0.179 0.289 LRRK2 9 1.293 0.280NLRP1 10, 11, 12, 13, 14 0.761 0.718 PMS1 15, 16, 17 0.461 0.284 MAK 180.201 0.309 CPD 19 1.294 0.220 CST7 20 0.740 0.238 DUSP5 21 1.219 0.241PTPRG 22 0.380 0.139 IL6R 23, 24 1.135 0.294 GHR 25 0.576 0.241 CSF3 26,27, 28 1.663 0.223 SPNS1 29 0.276 0.305 low temperature positive control1.000 0.754 rescue

Example 8 Analysis of the Trans-Epithelial Chloride Flux in CFTR ΔF508Homozygous Primary Bronchial Epithelial Cells

A further validation of the correction of ΔF508 CFTR is the correctionof trans-epithelial chloride transport in primary bronchial epithelialcell cultures from a CF patient, grown in a filter support. Thesewell-differentiated primary human bronchial epithelial cell culturesderived from CF patients homozygous for the ΔF508 CFTR mutation show aresidual forskolin and genistein-stimulated chloride flux that is lessthan 2% of non-CF control cell cultures. A standard drug additionprotocol (Devor et al., 2000) can be used to detect transepithelialcurrents due to CFTR. Short-circuit current (I_(sc)) across HBE primarycultures can be measured as described (Myerburg et al., 2006; Myerburget al., 2008). Cells cultured on filter supports are mounted in modifiedUssing chambers, and the cultures are continuously short-circuited withan automatic voltage clamp (Physiological Instruments, San Diego,Calif.). The basolateral bathing solution composition is: 120 mM NaCl,25 mM NaHCO₃, 3.3 mM KH₂PO₄, 0.8 mM K₂HPO₄, 1.2 mN MgCl₂, 1.2 mM CaCl₂,and 10 mM glucose. A basal-to-apical Cl gradient can be imposed byreducing the NaCl concentration of the apical bathing solution byreplacing NaCl with equimolar Na-gluconate. The chambers are maintainedat 37° C. and gassed continuously with a mixture of 95% O₂-5% CO₂ whichfixed the pH at 7.4. Following a 5 min equilibration period, thebaseline I_(sc) is recorded. Sodium currents are blocked by addition ofthe epithelial sodium channel (ENaC) blocker, amiloride (10 μM), to theapical bath. Subsequently, the cAMP agonist—forskolin (10 μM, Sigma),the CFTR potentiator—genistein (50 μM, Sigma), and the CFTR channelblocker—CFTRinh-172 (10 μM; Calbiochem, San Diego, Calif.) are addedsequentially, at the current steady-state, to determine cAMP-stimulatedCFTR currents. Addition of the CFTR inhibitor CFTRinh-172 is done toshow specificity of ion flux through CFTR (Ma et al., 2002). Using thisanalysis, the transepithelial currents associated with knockdown of thetargets are shown in Table 6. Each of these targets shows a significantincrease in chloride transport across the epithelial monolayer.Interestingly, the level of CFTR response can reach up to 20% ofwild-type CFTR-mediated currents, suggesting a clinically meaningfullevel of CFTR channel activity (Sheppard et al., 1993).

TABLE 6 Efficacy of the restoration of chloride transport in primary CFbronchial epithelial cell culture relevant to the presentexpression-inhibitory agent invention Forskolin-Genistein inducedchloride flux in CF primary TARGET Gene epithelial cells compared toSymbol SEQ ID NO: DNA control cells (non-CF) UGT3A2 2 22.9% PHGDH 3 6.6%B3GNT3 4 4.7% PPIH 5 9.1% CELSR3 6 11.8% MC2R 7 6.8% MAS1L 8 6.6% LRRK29 15.9% NLRP1 10, 11, 12, 13, 14 9.0% PMS1 15, 16, 17 9.3% MAK 18 9.5%CPD 19 14.8% CST7 20 10.7% DUSP5 21 15.5% PTPRG 22 10.7% IL6R 23, 2419.9% GHR 25 11.6% CSF3 26, 27, 28 9.4% SPNS1 29 13.4%

In the table above the knock-down sequence corresponding to SEQ 57 wasused, which demonstrates a specific effect with UGT3A2. However due tothe close homology and the high level of sequence identity betweenUGT3A2 and UGT3A1 it would be expected that a knock down of UGT3A1 wouldhave a similar effect on the restoration of chloride transport inprimary CF bronchial epithelial cells.

REFERENCES

-   Antonin, W., C. Holroyd, D. Fasshauer, S. Pabst, G. F. Von Mollard,    and R. Jahn. 2000. A SNARE complex mediating fusion of late    endosomes defines conserved properties of SNARE structure and    function. Embo J. 19:6453-64.-   Bilan, F., V. Thoreau, M. Nacfer, R. Derand, C. Norez, A.    Cantereau, M. Garcia, F. Becq, and A. Kitzis. 2004. Syntaxin 8    impairs trafficking of cystic fibrosis transmembrane conductance    regulator (CFTR) and inhibits its channel activity. J Cell Sci.    117:1923-35.-   Carolan, B. J., A. Heguy, B. G. Harvey, P. L. Leopold, B. Ferris,    and R. G. Crystal. 2006. Up-regulation of expression of the    ubiquitin carboxyl-terminal hydrolase L1 gene in human airway    epithelium of cigarette smokers. Cancer Res. 66:10729-40.-   Cheng, S. H., R. J. Gregory, J. Marshall, S. Paul, D. W.    Souza, G. A. White, C. R. O'Riordan, and A. E. Smith. 1990.    Defective intracellular transport and processing of CFTR is the    molecular basis of most cystic fibrosis. Cell. 63:827-34.-   Denning, G. M., M. P. Anderson, J. F. Amara, J. Marshall, A. E.    Smith, and M. J. Welsh. 1992. Processing of mutant cystic fibrosis    transmembrane conductance regulator is temperature-sensitive.    Nature. 358:761-4.-   Devor, D. C., Bridges, R. J., and Pilewski, J. M. (2000).    Pharmacological modulation of ion transport across wild-type and    DeltaF508 CFTR-expressing human bronchial epithelia. Am J Physiol    Cell Physiol 279, C461-479.-   Fischer, D. F., A. K. Scaffidi, S. Griffioen, M. Roseboom, and R. A.    Janssen. 2006. Identification of novel drug targets to treat cystic    fibrosis using adenoviral knock-down technology. Ped Pulmonol.    41:Suppl. 29, p. 209.-   Galietta, L. J., P. M. Haggie, and A. S. Verkman. 2001a. Green    fluorescent protein-based halide indicators with improved chloride    and iodide affinities. FEBS Lett. 499:220-4.-   Galietta, L. V., S. Jayaraman, and A. S. Verkman. 2001b. Cell-based    assay for high-throughput quantitative screening of CFTR chloride    transport agonists. Am J Physiol Cell Physiol. 281:C1734-42.-   Gruenert, D. C., M. Willems, J. J. Cassiman, and R. A.    Frizzell. 2004. Established cell lines used in cystic fibrosis    research. J Cyst Fibros. 3 Suppl 2:191-6.-   Guggino, W. B., and B. A. Stanton. 2006. New insights into cystic    fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell    Biol. 7:426-36.-   Hwang, T. C., F. Wang, I. C. Yang, and W. W. Reenstra. 1997.    Genistein potentiates wild-type and delta F508-CFTR channel    activity. Am J Physiol. 273:C988-98.-   Lambert, G., B. Becker, R. Schreiber, A. Boucherot, M. Reth, and K.    Kunzelmann. 2001. Control of Cystic Fibrosis Transmembrane    Conductance Regulator Expression by BAP31. J Biol. Chem.    276:20340-20345.-   Li, H., D. N. Sheppard, and M. J. Hug. 2004. Transepithelial    electrical measurements with the Ussing chamber. J Cyst Fibros. 3    Suppl 2:123-6.-   Ma, L., H. A. Bluyssen, M. De Raeymaeker, V. Laurysens, N. van der    Beek, H. Pavliska, A. J. van Zonneveld, P. Tomme, and H. H. van    Es. 2001. Rapid determination of adenoviral vector titers by    quantitative real-time PCR. J Virol Methods. 93:181-8.-   Ma, T., Thiagarajah, J. R., Yang, H., Sonawane, N. D., Folli, C.,    Galietta, L. J., and Verkman, A. S. (2002). Thiazolidinone CFTR    inhibitor identified by high-throughput screening blocks cholera    toxin-induced intestinal fluid secretion. J Clin Invest 110,    1651-1658.-   Myerburg, M. M., Butterworth, M. B., McKenna, E. E., Peters, K. W.,    Frizzell, R. A., Kleyman, T. R., and Pilewski, J. M. (2006). Airway    surface liquid volume regulates ENaC by altering the serine    protease-protease inhibitor balance: a mechanism for sodium    hyperabsorption in cystic fibrosis. J Biol Chem 281, 27942-27949.-   Myerburg, M. M., McKenna, E. E., Luke, C. J., Frizzell, R. A.,    Kleyman, T. R., and Pilewski, J. M. (2008). Prostasin expression is    regulated by airway surface liquid volume and is increased in cystic    fibrosis. Am J Physiol Lung Cell Mol Physiol 294, L932-941.-   Prince, L. S., R. B. Workman, Jr., and R. B. Marchase. 1994. Rapid    endocytosis of the cystic fibrosis transmembrane conductance    regulator chloride channel. Proc Natl Acad Sci USA. 91:5192-6.-   Quinton, P. M. 1990. Cystic fibrosis: a disease in electrolyte    transport. Faseb J. 4:2709-17.-   Riordan, J. R., J. M. Rommens, B. Kerem, N. Alon, R. Rozmahel, Z.    Grzelczak, J. Zielenski, S. Lok, N. Playsic, J. L. Chou, M. L.    Drumm, M. C. Ianuzzi, F. C. Collins, and L.-C. Tsui. 1989.    Identification of the cystic fibrosis gene: cloning and    characterization of complementary DNA. Science. 245:1066-73.-   Rowe, S. M., S. Miller, and E. J. Sorscher. 2005. Cystic fibrosis. N    Engl J Med. 352:1992-2001.-   Sheppard, D. N., Rich, D. P., Ostedgaard, L. S., Gregory, R. J.,    Smith, A. E., and Welsh, M. J. (1993). Mutations in CFTR associated    with mild-disease-form Cl-channels with altered pore properties.    Nature 362, 160-164.-   Thoreau, V., T. Berges, I. Callcbaut, Z. Guillier-Gencik, L.    Gressin, A. Bernheim, F. Karst, J. P. Mornon, A. Kitzis, and J. C.    Chomel. 1999. Molecular cloning, expression analysis, and    chromosomal localization of human syntaxin 8 (STX8). Biochein    Biophys Res Commun. 257:577-83.-   Ulloa-Aguirre, A., J. A. Janovick, S. P. Brothers, and P. M.    Conn. 2004. Pharmacologic rescue of conformationally-defective    proteins: implications for the treatment of human disease. Traffic.    5:821-37.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

From the foregoing description, various modifications and changes in thecompositions and methods of this invention will occur to those skilledin the art. All such modifications coming within the scope of theappended claims are intended to be included therein.

1. A method for identifying a compound that increases the functionalactivity of CF-associated mutant CFTR, comprising: (a) contacting acompound with a polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 31, 45, 30, 32-44, and 46-55 andfragments thereof; and (b) measuring a compound-polypeptide propertyrelated to CF-associated mutant CFTR activity.
 2. The method accordingto claim 1, wherein said polypeptide is in an in vitro cell-freepreparation.
 3. The method according to claim 1, wherein saidpolypeptide is present in a mammalian cell.
 4. The method of claim 2,wherein said property is a binding affinity of said compound to saidpolypeptide.
 5. The method of claim 1, wherein the method is used toidentify compounds that promote migration of ΔF508-CFTR to the plasmamembrane.
 6. The method of claim 4, which additionally comprises thesteps of: c) contacting a population of mammalian cells expressing saidpolypeptide with the compound that exhibits a binding affinity of atleast 10 micromolar; and d) identifying a compound that increases thefunctional activity of CF-associated mutant CFTR.
 7. The method of claim1, wherein said property is increased activity of ΔF508-CFTR or CFTR. 8.The method according to claim 1, wherein said property is the activityof said polypeptide.
 9. The method according to claim 1, wherein saidproperty is the expression of said polypeptide.
 10. The method accordingto claim 8, which additionally comprises the steps of: c) contacting apopulation of mammalian cells expressing said polypeptide with thecompound that significantly inhibits the expression or activity of thepolypeptide; and d) identifying the compound that increases thefunctional activity of CF-associated mutant CFTR.
 11. The methodaccording to claim 1, which additionally comprises the step of comparingthe compound to be tested to a control.
 12. The method according toclaim 11, wherein said control is where the polypeptide has not beencontacted with said compound.
 13. The method according to claim 6, whichadditionally comprises the step of comparing the compound to a control,wherein said control is a population of mammalian cells that does notexpress said polypeptide.
 14. The method according to claim 1, whereinsaid compound is selected from the group consisting of compounds of acommercially available screening library and compounds having bindingaffinity for a polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 31, 45, 30, 32-44, and 46-55.15. The method according to claim 1, wherein said compound is a peptidein a phage display library or an antibody fragment library.
 16. An agenteffective in increasing the functional activity of CF-associated mutantCFTR, selected from the group consisting of an antisense polynucleotide,a ribozyme, and a small interfering RNA (siRNA), wherein said agentcomprises a nucleic acid sequence complementary to, or engineered from,a naturally-occurring polynucleotide sequence of about 17 to about 30contiguous nucleotides of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 2, 19, 1, 3-18 and 20-29.
 17. The agentaccording to claim 16, wherein a vector in a mammalian cell expressessaid agent.
 18. The agent according to claim 16, which increases thefunctional activity of CF-associated mutant CFTR.
 19. The agentaccording to claim 17, wherein said vector is an adenoviral, retroviral,adeno-associated viral, lentiviral, a herpes simplex viral or asendaiviral vector.
 20. The agent according to claim 16, wherein saidantisense polynucleotide and said siRNA comprise an antisense strand of17-25 nucleotides complementary to a sense strand, wherein said sensestrand is selected from 17-25 continuous nucleotides of a nucleic acidsequence selected from the group consisting of SEQ ID NO: 2, 19, 1, 3-18and 20-29.
 21. The agent according to claim 20, wherein said siRNAfurther comprises said sense strand.
 22. The agent according to claim21, wherein said sense strand is selected from the group consisting ofSEQ ID NO: 56, 57, 81, 82, 58-80 and 83-99.
 23. The agent according toclaim 20, wherein said siRNA further comprises a loop region connectingsaid sense and said antisense strand.
 24. The agent according to claim23, wherein said loop region comprises a nucleic acid sequence selectedfrom the group consisting of UUGCUAUA or GUUUGCUAUAAC (SEQ ID NO: 100).25. The agent according to claim 16, wherein said agent is an antisensepolynucleotide, ribozyme, or siRNA comprising a nucleic acid sequencecomplementary to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 56, 57, 81, 82, 58-80 and 83-99.
 26. Apharmaceutical composition comprising a therapeutically effective amountof an agent according to claim 16 in admixture with a pharmaceuticallyacceptable carrier.
 27. A method for the treatment and/or prevention ofa disease involving ER-associated protein misfolding in a subjectsuffering from or susceptible to the disease, comprising administering atherapeutically effective amount of a pharmaceutical compositionaccording to claim
 26. 28. The method according to claim 27 wherein thedisease is selected from Cystic Fibrosis, Parkinson's disease, Gaucher'sdisease, nephrogenic diabetes insipidus, emphysema and liver disease(alpha-1-antitrypsin deficiency), Maple syrup urine disease, Fabry'sdisease, hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia,beta-galactosidosis, Wilson's disease, long QT syndrome, retinitispigmentosa, transthyretin-linked amyloidosis, Alzheimer's disease, priondisease, and inclusion body myositis.
 29. The method according to claim28, wherein the disease is Cystic Fibrosis.
 30. A method for thetreatment and/or prevention of a disease involving ER-associated proteinmisfolding, or the treatment or prevention of a condition characterizedby ER-associated protein misfolding, comprising administering atherapeutically effective amount of an agent according to claim
 16. 31.The method according to claim 30, wherein the disease is selected fromthe group consisting of Cystic Fibrosis, Parkinson's disease, Gaucher'sdisease, nephrogenic diabetes insipidus, emphysema and liver disease(alpha-1-antitrypsin deficiency), Maple syrup urine disease, Fabry'sdisease, hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia,beta-galactosidosis, Wilson's disease, long QT syndrome, retinitispimentosa, transthyretin-linked amyloidosis, Alzheimer's disease, priondisease, and inclusion body myositis.
 32. The method according to claim30, wherein the disease is Cystic Fibrosis.
 33. (canceled)
 34. A methodfor diagnosing a pathological condition in a subject involvingER-associated protein misfolding, or a pathological condition involvinginflammation, comprising determining a first amount or activity ofpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 31, 45, 30, 32-44, and 46-55 present in abiological sample obtained from said subject, and comparing said firstamount or activity with the ranges of amounts or activities of thepolypeptide determined in a population of healthy subjects, wherein anincrease of the amount or activity of polypeptide in said biologicalsample compared to the range of amounts or activities determined forhealthy subjects is indicative of the presence of the pathologicalcondition.
 35. A method for the treatment and/or prevention of a diseaseinvolving inflammation in a subject suffering from or susceptible to thedisease, comprising administering a therapeutically effective amount ofa pharmaceutical composition according to claim
 26. 36. The methodaccording to claim 35 wherein the disease is selected from CysticFibrosis, COPD and asthma.
 37. A method for the treatment and/orprevention of a disease involving or characterized by inflammation,comprising administering a therapeutically effective amount of an agentaccording to claim
 16. 38. The method according to claim 37, wherein thedisease is selected from the group consisting of Cystic Fibrosis, COPDand asthma.
 39. (canceled)
 40. (canceled)